JP2003508350A - Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components - Google Patents

Abstract

A method for protecting a peptide from peptidase activity in vivo, the peptide being composed of between 2 and 50 amino acids and having a C-terminus and an N-terminus and a C-terminus amino acid and an N-terminus amino acid is described. In the first step of the method, the peptide is modified by attaching a reactive group to the C-terminus amino acid, to the N-terminus amino acid, or to an amino acid located between the N-terminus and the C-terminus, such that the modified peptide is capable of forming a covalent bond in vivo with a reactive functionality on a blood component. In the next step, a covalent bond is formed between the reactive group and a reactive functionality on a blood component to form a peptide-blood component conjugate, thereby protecting said peptide from peptidase activity. The final step of the method involves the analyzing of the stability of the peptide-blood component conjugate to assess the protection of the peptide from peptidase activity.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to modified therapeutic peptides. In particular, the invention relates to the protection of endogenous therapeutic peptides from peptidase activity by a modification that allows the peptides to be selectively conjugated to blood components, thus protecting the peptides from peptidase activity, And to increase the duration of action of therapeutic peptides for treating various disorders.

BACKGROUND OF THE INVENTION Many endogenous peptides have been described as being major components of biological processes. Some of these peptides have been identified as major therapeutic agents for the management of various disorders. In general, endogenous peptides are more desirable as therapeutic agents than synthetic peptides having non-natural sequences. Because endogenous peptides do not generate an immune response due to their intrinsic properties. further,
The endogenous peptide is highly specific for its target receptor and is easy to synthesize and manufacture. However, the major difficulty in delivering such therapeutic peptides is their short serum half-life, which is primarily due to rapid serum clearance and proteolytic degradation via the action of peptidases.

Peptidases break peptide bonds in peptides by inserting a water molecule across the bond. In general, most peptides are destroyed by peptidases in the body in a matter of minutes or less. Moreover, some peptidases are specific for certain types of peptides, making their degradation even faster. Therefore, when the peptide is used as a therapeutic agent, its activity is generally diminished. This is because this peptide is rapidly degraded in the body due to the action of peptidases.

One way to overcome this drawback is to administer a large dosage of the therapeutic peptide of interest to the patient, whereby it is therapeutically effective, even if some of the peptide is degraded. It's about being enough to be there. However, this method is very uncomfortable for the patient. Since most therapeutic peptides cannot be administered orally, the therapeutic peptide must be constantly infused, must be given frequently by intravenous injection, or is frequently given by the inconvenient route of subcutaneous injection. Either must be done. The need for frequent administration also results in many potential peptide therapeutics having unacceptably high planned costs per course of treatment. The presence of large amounts of degraded peptide can also give rise to unwanted side effects.

Discomfort and high cost of administration are two reasons why most therapeutic peptides with attractive bioactivity profiles are not developed as drug candidates. Instead, these therapeutic peptides are used as templates for the development of peptidomimetic compounds that replace therapeutic peptides. Biotechnology and large pharmaceutical companies frequently undertake long and expensive optimization programs to develop non-peptidic organic compounds that mimic the activity seen in therapeutic peptides without incurring unacceptable side effect profiles. Try to do. For example, cyclic peptides, peptidomimetics and expensive SAR (structure-activity relationship) and small molecules resulting from molecular modeling studies have led to the development of numerous peptidomimetics. However, these peptidomimetics do not, in any way, reflect the exact initial biological properties of the therapeutic peptide and are thus inferior to endogenous therapeutic peptides as therapeutic agents.

An alternative to creating peptidomimetics is to block the activity of peptidases to prevent degradation of the therapeutic peptide, or to slow degradation of the therapeutic peptide while maintaining biological activity. Modifying the therapeutic peptide in the manner described above. Such methods include conjugation with polymeric materials (eg, dextran, polyvinylpyrrolidone, glycopeptides, polyethylene glycol and polyamino acids), conjugation with adroitin sulfate, and polysaccharides, low molecular weight compounds. (For example, aminolecithin
icin), fatty acids, vitamin B ₁₂ , and glycosides). However, these conjugates are still often sensitive to protease activity. Moreover, the therapeutic activity of these peptides is often diminished by the addition of polymeric material. Finally, there is a risk that these conjugates will generate an immune response when the material is injected in vivo. Some methods are
Conjugating with a carrier protein ex vivo, resulting in the generation of random conjugates. Since conjugates are difficult to manufacture, their interest is limited by the commercial availability of carriers and their poor pharmacoeconomics.

Therefore, therapeutic peptides have been modified to protect them from peptidase activity,
And there is a need for new methods to provide a longer duration of action in vivo while maintaining low toxicity while still retaining the therapeutic benefit of the modified peptides.

SUMMARY OF THE INVENTION The present invention provides for the modification of a therapeutic peptide of interest and attachment of this peptide to a protein carrier such that the action of peptidase is prevented or slowed. It relates to overcoming the problem of degradation of peptides in the body. More particularly, the present invention relates to novel chemically reactive derivatives of therapeutic peptides, especially therapeutic peptide-maleimide derivatives, which are capable of reacting with available functional groups of blood proteins to form covalent bonds. . The invention also relates to novel chemically reactive derivatives or analogs of such therapeutic peptides. The present invention is
Furthermore, it relates to the therapeutic use of such compounds.

The present invention utilizes synthetic modifications to the first residue to be cleaved to modify the therapeutic peptide and to a protein carrier (preferably albumin) via in vivo or ex vivo techniques. Attached to prevent or reduce the action of peptidases. Therapeutic peptides typically include N-terminal portion, C-terminal portion,
Alternatively, it is active in a portion inside the peptide chain. Using the techniques of the present invention, sites other than the active portion of the therapeutic peptide are modified with specific reactive groups. These reactive groups may form covalent bonds with functional groups present on blood components. The reactive group is located at a site such that when the therapeutic peptide binds to blood components, the peptide retains a significant percentage of the parent compound's activity.

The modification of the therapeutic peptide by the chemical modification used in the present invention is done in such a way that all or most of the peptide specificity is preserved despite attachment to blood components. . The therapeutic peptide-blood component complex is now able to translocate to various body regions without being degraded by peptidases and while the peptide still retains its therapeutic activity. The present invention is applicable to all known therapeutic peptides and can be easily tested under physiological conditions by directly comparing the pharmacokinetic parameters for free peptides and modified therapeutic peptides. It

The present invention relates to modified therapeutic peptides which are capable of forming a peptidase-stabilized therapeutic peptide consisting of between 3 and 50 amino acids. The peptide has a carboxy-terminal amino acid, an amino-terminal amino acid, a therapeutically active region of amino acids and a less therapeutically active region of amino acids. This peptide reacts with amino, hydroxyl, or thiol groups on blood components to form a stable covalent bond, thereby forming a therapeutic peptide stabilized against peptidases. Including a group. In the peptides of the invention, the reactive group is selected from the group consisting of a succinimidyl group and a maleimide group, and the reactive group is attached to an amino acid located in the therapeutically less active region of the amino acid.

In one embodiment, the therapeutically active region of the peptide comprises the carboxy terminal amino acid and the reactive group is attached to the amino terminal amino acid.

In another embodiment, the therapeutically active region of the peptide comprises the amino terminal amino acid and the reactive group is attached to the carboxy terminal amino acid.

In another embodiment, the therapeutically active region of the peptide comprises a carboxy terminal amino acid and the reactive group is attached to an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid.

In yet another embodiment, the therapeutically active region comprises an amino terminal amino acid and the reactive group is attached to an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid.

The present invention also relates to methods of synthesizing modified therapeutic peptides. This method includes the following steps. In the first step, if the therapeutic peptide does not contain cysteine, the peptide is synthesized from the carboxy terminal amino acid and a reactive group is added to the carboxy terminal amino acid. Alternatively, a terminal lysine is added to the carboxy terminal amino acid and a reactive group is added to this terminal lysine. In the second step, if the therapeutic peptide contains only one cysteine,
The cysteine is reacted with a protecting group, which then adds a reactive group to an amino acid in the therapeutically less active region of the peptide. In the third step, if the therapeutic peptide contains two cysteines as disulfide bridges, these two cysteines are oxidized to form a reactive group at the amino terminal amino acid of the therapeutic peptide,
Alternatively, it is added to the carboxy-terminal amino acid or an amino acid located between the carboxy-terminal amino acid and the amino-terminal amino acid. In the fourth step, if the therapeutic peptide contains more than two cysteines as disulfide bridges, these cysteines are sequentially oxidized at the disulfide bridges and the peptide is purified, after which reactive groups are removed. Add to the carboxy terminal amino acid.

The present invention also relates to a method for protecting a therapeutic peptide in vivo from peptidase activity, which peptide consists of between 3 and 50 amino acids and is carboxy-terminal and amino-terminal, as well as a carboxy-terminal amino acid. And having an amino terminal amino acid. The method comprises the steps of: (a) attaching a reactive group to a carboxy-terminal amino acid, an amino-terminal amino acid, or an amino acid located between the amino-terminal amino acid and the carboxy-terminal amino acid to form a peptide. A step of modifying so that the modified peptide is capable of forming a covalent bond in vivo with a reactive functional group of a blood component; (b) a reactive group and a reactive functional group of a blood component. Forming a peptide-blood component conjugate with a peptide-blood component conjugate thereby protecting the peptide from peptidase activity; and (c) the peptide-blood component conjugate. Analyzing the stability of the peptide to assess the protection of this peptide from peptidase activity. These steps may be performed in vivo or ex vivo.

The invention also relates to a method for the protection of a therapeutic peptide in vivo from peptidase activity, which peptide consists of between 3 and 50 amino acids, and the therapeutically active region of amino acids and amino acids. Has a therapeutically lower active region of. The method comprises the following steps: (a) determining the therapeutically active region of the amino acid; (b) attaching the reactive group to the amino acid to form a modified peptide, thereby forming the amino acid. Altering the peptide at an amino acid contained in the therapeutically less active region of, so that the modified peptide has therapeutic activity and is shared in vivo with the reactive functional group of the blood component. Can form a bond,
Step (c) forming a covalent bond between the reactive entity and the reactive functional group of the blood component to form a peptide-blood component conjugate, whereby the peptide is separated from the peptidase activity. Protecting; and (d) analyzing the stability of the peptide-blood component conjugate to assess protection of the peptide from peptidase activity. These steps may be performed in vivo or ex vivo.

Peptides useful in the compositions and methods of the present invention include, but are not limited to, the peptides set forth in SEQ ID NO: 1 through SEQ ID NO: 1617.

DETAILED DESCRIPTION OF THE INVENTION Definitions To ensure a full understanding of the invention, the following definitions are provided: Reactive group: A reactive group is an entity that can form a covalent bond. Is. Such reactive groups are linked or attached to the therapeutic peptide of interest. The reactive group is generally stable in an aqueous environment and is usually a carboxy group, a phosphoryl group, or a conventional acyl group, either present as an ester or mixed anhydride, or as an imidate, whereby An amino group at the target site of mobile blood components,
It may form covalent bonds with functional groups such as hydroxy or thiol. To a large extent, these esters include phenolic compounds or are thiol esters, alkyl esters, phosphate esters and the like. Reactive groups include succimidyl and maleimide groups.

Functional group: A functional group is a group on a blood component with which a reactive group of a modified therapeutic peptide reacts to form a covalent bond, including mobile and fixed proteins. Functional groups typically include hydroxyl groups for attachment to ester-reactive groups, thiol groups for attachment to maleimide, imidate and thiol ester groups; activated carboxyl groups, phosphoryl groups or other groups on reactive groups. It includes an amino group for attachment to any acyl group.

Blood component: The blood component may be fixed or movable.
Fixed blood components are non-moving blood components and are tissues, membrane receptors, intervening proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and the membrane and membrane receptors to which they bind, body cells. (Somat
ic body cells), skeletal and smooth muscle cells, neural components, osteocytes and osteoclasts and all body tissues, especially those associated with the circulatory and lymphatic systems. A mobile blood component is a blood component that does not have a fixed position for any extended period of time (typically no more than 5 minutes, more usually 1 minute). These blood components are not membrane bound and are present in blood for an extended period of time and are present at a minimum concentration of at least 0.1 μg / ml.
Mobile blood components include serum albumin, transferrin, ferritin, and immunoglobulins such as IgM and IgG. The mobile blood component has a half-life of at least about 12 hours.

Protecting Group: A protecting group is a chemical moiety utilized to protect a peptide derivative from reacting with itself. Various protecting groups are described in US Pat. No. 5,493,
No. 007, which is incorporated herein by reference. Such protecting groups include acetyl, fluorenylmethyloxycarbonyl (Fmo
c), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (C
bz) and the like. The specific protected amino acids are shown in Table 1.

Linking Group: A linking group is a chemical moiety that links or connects a reactive group to a therapeutic peptide. The linking group is one or more alkyl groups, alkoxy groups, alkenyl groups, alkynyl groups or amino groups substituted with alkyl groups, cycloalkyl groups, polycyclic groups, aryl groups, polyaryl groups, substituted aryl groups, heterocycles. Formula groups and substituted heterocyclic groups can be included. The linking group may also include a polyethoxy amino acid, such as AEA ((2-amino) ethoxyacetic acid) or the preferred linking group AEEA ([2- (2-amino) ethoxy)] ethoxyacetic acid). Preferred linking groups are
It is aminoethoxyethoxyacetic acid (AEEA).

Sensitive Functional Group: A sensitive functional group is a group of atoms that provides a potential reactive site for a therapeutic peptide. When present, sensitive functional groups can be selected as attachment points for linker-reactive group modification. Sensitive functional groups include, but are not limited to, carboxyl groups, amino groups, thiol groups, and hydroxyl groups.

Modified Therapeutic Peptide: A modified therapeutic peptide is a therapeutic peptide modified by the attachment of a reactive group and is stabilized against peptidases by conjugation with blood components. Formed peptides can be formed. Reactive groups can be attached to therapeutic peptides either through a linking group or optionally without a linking group. It is also contemplated that one or more additional amino acids may be added to the therapeutic peptide to facilitate attachment of reactive groups. The modified peptides can be administered in vivo such that conjugation with blood components occurs in vivo, or they are first conjugated in vitro with blood components and then to the resulting peptidase. The stabilized peptide (as defined below) can be administered in vivo. The terms "modified therapeutic peptide" and "
"Modified peptide" may be used interchangeably herein.

Therapeutic peptide stabilized against peptidase: A therapeutic peptide stabilized against peptidase is a functional group of a modified peptide and a blood component with or without a linking group. It is a modified peptide conjugated to a blood component via a covalent bond formed with a group. Peptidase stabilized peptides are more stable in vivo in the presence of peptidases than non-stabilized peptides. Therapeutic peptides stabilized against peptidases generally have at least 10 peptides compared to unstabilized peptides of the same sequence.
It has a half-life increased by -50%. Peptidase stability is determined by comparing the half-life of the unmodified therapeutic peptide in serum or blood with the half-life of the corresponding modified therapeutic peptide in serum or blood. Half-life is determined by sampling serum or blood after administration of the modified and unmodified peptides and determining the activity of the peptide. In addition to determining activity, the length of therapeutic peptides can also be measured.

(Therapeutic peptide) -A therapeutic peptide, as used in the present invention, is an amino acid chain between 2 and 50 amino acids having therapeutic activity as defined below. . Each therapeutic peptide has an amino terminus (also called the N-terminal or amino-terminal amino acid), a carboxyl terminus (also called the C-terminal or carboxyl-terminal amino acid), and an internal amino acid located between the amino and carboxyl termini. This amino terminus is defined by the only amino acid in the therapeutic peptide that has a free α-amino group. This carboxyl end is
It is defined by the only amino acid in its therapeutic peptide that has a free α-carboxyl group.

The therapeutic peptides used in the present invention generally include a therapeutically active region located at their amino termini, carboxyl termini, or internal amino acids. This therapeutically active region is blind (bli), as defined in more detail herein.
nd) substitutions or structure activity relationship (SAR) driven substitutions. S
AR is an analysis that defines the relationship between the structure of a molecule and its pharmacological activity for a series of compounds. Alternatively, where the therapeutically active region has been previously defined and is available in the literature, reference to a reference such as a scientific journal provides access to the therapeutically active region. obtain. Knowledge of the location of the therapeutically active region of the peptide is important for modifying the therapeutic peptide, as defined in more detail below.

The therapeutic peptides used in the present invention also have a therapeutically less active region generally located at or near the amino terminus, the carboxyl terminus or near the carboxyl terminus, or at or near an internal amino acid. Including. This therapeutically less active region is a region of amino acids that is inconsistent with the therapeutically active region of the therapeutic peptide. This therapeutically less active region is generally located away from the therapeutically active region so that modification in that therapeutically less active region results in therapeutic activity of the therapeutic peptide. Has virtually no effect on For example, if the therapeutically active region is located at the amino terminus, the therapeutic peptide will be modified at either the carboxyl terminus or an internal amino acid. Alternatively, if the therapeutically active region is located at the carboxyl terminus, then the therapeutic peptide is modified at either the amino terminus or an internal amino acid. Finally, if the therapeutically active region is located in the internal region, the therapeutic peptide is modified at either the amino or carboxyl termini.

“Therapeutic activity” is any activity associated with curing or treating a biological disorder in a patient. Examples of the therapeutic peptides include: pituitary hormones (eg, vasopressin, oxytocin, melanocyte stimulating hormone, adrenocorticotropic hormone, growth hormone); hypothalamic hormone (
For example, growth hormone releasing factor, adrenocorticotropic hormone releasing factor, prolactin releasing peptide, gonadotropin releasing hormone and its related peptides, luteinizing hormone releasing hormone, thyroid stimulating hormone releasing hormone, orexin, and somatostatin); thyroid hormone (for example, , Calcitonin, calcitonin precursors, and calcitonin gene-related peptides); parathyroid hormone and its related proteins; pancreatic hormones (eg, insulin and insulin-like peptides,
Glucagon, somatostatin, pancreatic polypeptide, amylin, peptide YY, and neuropeptide Y); digestive hormones (eg, gastrin, gastrin releasing peptide, gastrin inhibitory peptide, cholecystokinin, secretin, motilin, and vasoactive intestinal peptide); Natriuretic peptides (eg, atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide); neurokinins (neurokinin A, neurokinin B and P
Substances); renin-related peptides (eg renin substances and inhibitors and angiotensin); endothelins (including big endothelin, endothelin A receptor antagonists and saraphotoxin peptides); and other peptides (eg adrenomedullin peptides,
Allatostatin peptides, amyloid β protein fragments, antibiotic and antifungal peptides, apoptosis-related peptides, bag cell peptides, bombesin, bone Gla protein peptides, CA
RT peptide, chemotactic peptide, cortisstatin
Peptides, fibronectin fragments and fibrin-related peptides, FM
RF and analog peptides, galanin and related peptides, growth factors and related peptides, G therapeutic peptide-binding protein fragments, guanylin and uroguanylin, inhibin peptides, interleukins and interleukin receptor proteins, laminin fragments, leptin fragment peptides, leucokinin , Mast cell degranulation peptide, pituitary adenylate cyclase activating polypeptide, pancreatatin, peptide T
, Polypeptides, virus-related peptides, signaling reagents, toxins, and various peptides such as: adjuvant peptide analogs, alpha-mating factor, antiarrhythmic peptides, antifreeze polypeptides, anorexia-inducing peptides, bovine pineal gland Anti-reproductive peptide, brucine, C3 peptide P16, tumor necrosis factor, cadherin peptide, chromogranin A fragment, contraceptive tetrapeptide, conantokine (co
antokkin G, conantokin T, crustacean heart-acting peptide, C-telopeptide, cytochrome b588 peptide, decorsin, delicateous peptide, Δ-sleep inducing peptide, diazempam binding inhibitor fragment, monoxide. Nitrogen synthase blocking peptide, OVA peptide, platelet calpain inhibitor (P1), plasminogen activator inhibitor 1, lysine (rigin
), Schizophrenia-related peptide, serum thymus factor, sodium potassium A treatment peptidase inhibitor 1, speract, sperm activation peptide,
Systemin, thrombin receptor agonist, thymic γ2 factor, thymopentin, thymosin α1, thymic factor, tufucin, adipokinin hormone, uremic pentapeptide, and other therapeutic peptides).

In view of these definitions, the focus of the present invention is to modify the therapeutic peptide in vivo to protect it from peptidase activity, thereby comparing the administration of the peptide itself to a patient. To extend the effective therapeutic life of the therapeutic peptide.

1. Therapeutic Peptides Used in the Present Invention Peptide fragments selected from the determined amino acid sequences of therapeutic peptides as provided in the attached sequence listing (SEQUENCE LISTING) include the present invention. Constitutes the starting point in. This peptide ranges from 2 to 50 amino acids in length. The interchangeable terms "peptide fragment" and "peptide portion" are intended to include both synthetic and naturally occurring amino acid sequences derivable from naturally occurring amino acid sequences.

In one embodiment, peptides and peptide fragments are synthesized by conventional means, either by the bench-top method, or by an automated peptide synthesizer, as discussed in detail below. It However, it is also possible to obtain fragments of the peptide by fragmenting the naturally occurring amino acid sequence using, for example, proteolytic enzymes. In addition, Maniatis, T .; Et al., Molecular Biology: A
Laboratory Manual, Cold Spring Harbor
, New York (1982), incorporated herein by reference, through the use of recombinant DNA technology to obtain the desired fragment of the therapeutic peptide. The use of other new modifications to existing methodologies is also contemplated.

The present invention includes peptides derivable from the naturally occurring sequences of therapeutic peptides. If the peptide can be obtained by fragmenting a naturally occurring sequence, or based on knowledge of the sequence of the naturally occurring amino acid sequence or of the genetic material (DNA or RNA) encoding this sequence, A peptide is said to be "derivatizable from naturally occurring amino acid sequences" if it can be synthesized. Molecules that are said to be "derivatives" of peptides are included within the scope of this invention. Such a "derivative" has the following characteristics: (1) shares substantial homology with the therapeutic peptide or a similarly sized fragment of this peptide,
And (2) it may function with the same therapeutic activity as this peptide.

The amino acid sequence of the derivative of the peptide is at least 80% of that of either the peptide or the fragment of the peptide having the same amino acid residues as the derivative.
, And more preferably at least 90%, and most preferably at least 9%
A derivative of the peptide is said to share "substantial homology" with this peptide if they are 5% identical.

Derivatives of the invention, in addition to containing a sequence that is substantially homologous to the sequence of a naturally occurring therapeutic peptide, as discussed in detail below, have amino-terminal and / or
Or a fragment that may contain one or more additional amino acids at the carboxy terminus. Accordingly, the present invention relates to polypeptide fragments of therapeutic peptides that may include one or more amino acids that may not be present in the naturally occurring therapeutic peptide sequence, provided that such fragment is the therapeutic peptide. If the therapeutic activity exceeds the activity of.

Similarly, the invention includes a sequence that is substantially homologous to the sequence of a naturally occurring therapeutic peptide, but one of the amino and / or carboxy termini naturally found in that therapeutic peptide. Included are polypeptide fragments capable of deleting the above additional amino acids. Accordingly, the present invention relates to polypeptide fragments of therapeutic peptides that may lack one or more amino acids normally found in their naturally occurring peptide sequences, provided that such polypeptide is active in that therapeutic peptide. When it has a therapeutic activity of more than).

The present invention also includes obvious or trivial variants of the above fragments having non-significant amino acid substitutions (thus having an amino acid that differs from the amino acid sequence of its native sequence), provided that That variant has an activity that is substantially the same as the activity of the above derivative). Examples of explicit or trivial substitutions are: basic residues to each other (ie Lys to Arg), hydrophobic residues to each other (ie Ile to Leu) or aromatic residues to each other. (That is, Tyr to Phe), and the like.

As is known in the art, an amino acid residue can be in its protected or unprotected form with a suitable amino or carboxyl protecting group, as discussed in detail below. The variable length peptide can be in the form of the free amine (to the N-terminus), or its acid addition salt. Common acid addition salts are hydrohalic acid (hy
drohalic acid) salt (ie HBr, HI or more preferably HCl). Useful cations are alkali metal or alkaline earth metal cations (ie Na, K, Li, Ca, Ba, etc.) or amine cations (ie tetraalkylammonium, trialkylammonium (where alkyl is C _1- ). C ₁₂ ))).

Any peptide having therapeutic activity can be used in the present invention. The following list of peptides provides examples of peptides that can be used in the present invention. However, this example is not exclusive and in no way limits the number or type of peptides that can be used in the present invention. These therapeutic peptides and fragments produced from these peptides can be modified according to the invention and used therapeutically in the body.

(A. Pituitary Hormone (SEQ ID NOS: 1 to 72)) (Adrenocorticotropic Hormone (ACTH, or Corticotropin) (SEQ ID NOS: 1 to 22) -The endocrine function of the adrenal cortex is the anterior pituitary hormone ACTH) ACTH, a 39 amino acid peptide, is produced in corticotrophs of the anterior pituitary under the control of corticotropin-releasing factor ACTH from a 241 amino acid precursor known as pro-opiomelanocortin (POMC). Induced by post-translational modification.

The biological role of ACTH is to maintain the volume and viability of the adrenal cortex and to stimulate the production of corticosteroids (mainly cortisol and corticosterone). The mechanism of action of ACTH involves binding to the ACTH receptor followed by activation of adenylate cyclase, elevated cyclic AMP (cAMP), and increased protein kinase A (PKA) activity in adrenal cortex tissue. The major effect of these events is to increase the activity of side chain scission enzymes, which convert cholesterol to pregnenolone. Due to the distribution of the enzyme in various compartments of the adrenal cortex, the main physiological effect of ACTH is the production of glucocorticosteroids.

In addition to its ability to control adrenal cortical activity, ACTH appears to have diverse biological roles, including endocrine and endocrine regulation, temperature regulation, and effects on nerve regeneration and development. . In addition, ACTH and its fragments influence motivation, learning and behavior. Therefore, the use of ACTH as a therapeutic factor may help control these functions. Release of ACTH from the anterior pituitary is mediated by corticotropin releasing factor (CRF).

Growth Hormone Peptides (SEQ ID NOS: 23-24, 45) -Human placental lactogen (hPL), growth hormone, and prolactin (PrI) make up the growth hormone family, all about 200 amino acids and two Each has a disulfide bond and no glycosylation, each with specific receptors and unique characteristics for its activity, but all possess growth-promoting and mammary stimulatory activities. (22,000 daltons) is synthesized as a single polypeptide chain in eosinophilic pituitary somatotropin-producing cells.
Due to splicing, small amounts of smaller and smaller molecular forms are also secreted.

There are numerous genetic defects associated with GH. GH-deficient dwarfs lack the ability to synthesize or secrete GH, and these short stature individuals respond well to GH therapy. Pygmy lacks the IGF-1 response to GH but not its metabolic effects; therefore, in pygmy, the defect is essentially post-receptor (pos).
t-receptor)). Overall, Laron dwarfs have normal or excess plasma GH, but lack liver GH receptors, and low levels of circulating IGF-1. The defect in these individuals is due to the production of IGF-1
Closely related to the inability to respond to H. Excessive GH production before epiphyseal closure of long bones leads to macrosomia and, if GH becomes excessive after epiphyseal closure, terminal bone growth leads to the characteristic feature of acromegaly . Using GH as a therapeutic factor may help treat these disorders and possibly stimulate growth in other types of short stature with low or normal GH levels.

Melanocyte Stimulating Hormone (MSH) (SEQ ID NOs: 25-39) -Melanocyte stimulating hormone (MSH) is produced in the mid-pituitary under the control of dopamine. MSH may have important physiological roles in the regulation of vertebrate pigment cell melanin production, learning and behavioral neural functions, and fetal development. Sawyer, T .; K. Et al., Proc. Nat. Acad. Sci. USA,
79, 1751 (1982).

(Oxytocin (SEQ ID NOS: 40-44))-Oxytocin is involved in enhancing lactation, contracting the uterus, and prenatal pelvic laxity. Oxytocin secretion in lactating women is stimulated by direct neural feedback obtained by stimulation of the nipple during feeding. Physiological effects of oxytocin include contraction of mammary myoepithelial cells (inducing ejection of milk from the mammary gland) and stimulation of uterine smooth muscle contraction that encourages childbirth. Oxytocin causes myoepithelial cells surrounding the secretory acini of the mammary gland to contract, pushing milk through the duct. In addition, oxytocin stimulates the release of prolactin. And prolactin is a nutrition of the breast (trophi
c), and stimulates acini formation in milk. Thus, conjugated oxytocin can be used to assist lactation and to help relax the pelvis prior to birth. This oxytocin is also postpartum
Used to prevent uterine bleeding.

[0049]   Vasopressin (ADH) (SEQ ID NOS: 46-72) -vasopressin also has an
It is also known as urinary hormone (ADH). Because vasopressin is immersed in body fluids
It is a major regulator of permeability and leads to antidiuresis and increases blood pressure
Is. Vasopressin binds to plasma membrane receptors and transduces G proteins.
Acting cyclically, cyclic AMP / protein kinase A (cAMP / PKA)
Activates the regulatory system. The secretion of vasopressin is dependent on the osmotic receptor
is regulated in the hypothalamus. Osmotic receptors are used to detect water concentration.
Knowing, and increasing plasma osmolality stimulates an increase in vasopressin secretion. Minute
Excited vasopressin increases the rate of water reabsorption in tubular cells ⁺ Causes excretion of urine that is concentrated and thus a net decrease in osmotic pressure of body fluids
cause. Vasopressin deficiency is a condition known as diabetes insipidus.
It results in watery urine and polydipsia. Therefore, the combination
The use of derivatized vasopressin or vasopressin fragments eliminates these obstacles.
Prevents and allows regular maintenance of the body's osmotic pressure.

(B. Hypothalamic hormone (releasing factor)) (Corticotropin releasing factor (CRF) and related peptides (SEQ ID NOs: 73 to 73)
102))-41 amino acid peptide, corticotropin-releasing hormone (
CRF) plays an important role in coordinating the overall response to stress through actions in both the brain and the periphery. In the brain, CRF is mainly produced and secreted by small cell neurons in the paraventricular nucleus of the hypothalamus. From there, C
RF-containing neurons project into the portal capillaries of the median eminence and stimulate the secretion of adrenocorticotropic hormone (ACTH), β-endorphin, and other pituitary-derived proopiomelanocortin (POMC) -derived peptides. work. Subsequent ACTH-induced release of the adrenal glucocorticoids represents the final step in the hypothalamus-pituitary-adrenal system (HPA), which mediates the endocrine response to stress.
In addition to the neuroendocrine role of CRF, CRF also functions as a neurotransmitter and neuromodulator, exerting a broad spectrum of autonomic effects, behavior on physiological, pharmacological and pathological stimuli. Induces effects and immune effects.

Clinical studies have shown that CRF hypersecretion is associated with various diseases such as major depression, anxiety-related diseases, eating disorders, and inflammatory disorders. Low levels of CRF have been found in Alzheimer's disease, dementia, obesity and many endocrine disorders. Thus, the use of CRF as a therapeutic agent to counter the effects associated with high or low levels of CRF provides the basis for the treatment of diseases associated with abnormal CRF levels. Several peptide and non-peptide antagonists have been developed and extensively studied. This includes α-helix CRF (9-41), Astressin, D-PheCRF.
(12-41) (peptide antagonists) and CP-154526 (non-peptide antagonists). These CRF antagonists may provide novel agents for the treatment of depression, anxiety and other CRH related diseases. Thus, the conjugated CRF peptide can be used to maintain adrenal status and viability during long-term steroid use, or as an anti-inflammatory drug.

(Gonadotropin-releasing hormone-related peptide (GAP)
(SEQ ID NOS: 103-110) -GAP is a gonadotropin-releasing hormone (Gn
RH) included in the precursor molecule. GAP has prolactin inhibiting properties.
Gn-RH is a hormone secreted by the hypothalamus. It stimulates the release of gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). Low levels of circulating sex hormones reduce feedback inhibition of GnRH synthesis, leading to elevated levels of FSH and LH. The latter peptide hormone binds to gonadal tissue and results in sex hormone production via cyclic AMP (cAMP) and protein kinase A (PKA) mediated pathways. Conjugated G
nRH can be used to assist fertility or as a contraceptive agent in either males or females. This factor has applications in animals and humans.

Growth Hormone Releasing Factor (GRF) (SEQ ID NOS: 111-134) GRF is a hypothalamic peptide that plays an important role in controlling growth hormone synthesis and secretion in the anterior pituitary. Several structurally unrelated short peptides were also reported to induce growth hormone secretion by different mechanisms.

Under the influence of GRF, growth hormone is released into the systemic circulation and IGF is delivered to target tissues.
-1 is secreted. Growth hormone also has other more direct metabolic effects;
This is both a blood sugar raising effect and a lipolytic effect. The main source of systemic IGF-1 is the liver, but it also secretes most other tissues and systemic IGF-1.
Contribute to 1. Liver IGF-1 is considered to be the major regulator of tissue growth. In particular, IGF-1 secreted by the liver synchronizes growth throughout the whole body and results in a homeostatic ba of tissue size and mass.
lance). IGF-1 secreted by surrounding tissues is
It is generally considered to be an autocrine or paracrine in biological action. The use of conjugated GRF as a therapeutic agent to increase GH release then aids in the treatment of disorders involving GRF-regulated growth function.

(Luteinizing Hormone-Releasing Hormone (LH-RH) (SEQ ID NOS: 135-161)
) Luteinizing hormone-releasing hormone is gonadotropin, luteinizing hormone (
LH) and follicle-stimulating hormone (FSH) are important mediators in neuromodulation of secretion. LH-RH can alter sexual behavior by regulating plasma gonadotropin and sex steroid levels. Vale, W.A. W. ,
Peptides, Structure and Biological Fu
nction, Proceedings of the Six Amer
ican Peptide Symposium, Gross, E and Mei
enhofer, M .; Eds., 781 (1979). LH conjugated
-RH agents may be used to stimulate ovulation in humans or animals as an aid to fertility.

Orexin (SEQ ID NOS: 162-164) Orexin was recently discovered and
It is a family of characterized hypothalamic neuropeptides. Orexins stimulate appetite and food consumption. The genes are bilaterally and symmetrically expressed in the lateral hypothalamus. The outer part of the hypothalamus was early determined to be the "feeding center" of the hypothalamus. In contrast, the so-called satiety center is the ventromedial hypothalamus (v
is expressed in the endometrial hypothalamus) and is dominated by the leptin-regulated neuropeptide network.

(Prolactin-releasing peptide (SEQ ID NOS: 65 to 170)) Prolactin is
It is produced by the eosinophilic pituitary lactotrope. The prolactin-releasing peptide acts on the lactotrope to release prolactin. RPL initiates and maintains lactation in mammals (but usually only in estrogenic hormone-primed mammary tissue). The conjugated PRP can be used to increase lactation in humans or animals.

(Somatostatin (SEQ ID NOS: 171-201)) Somatostatin, also known as growth hormone release inhibitor (GIF), is a 14 amino acid peptide,
It is then secreted by both hypothalamic and pancreatic d cells (the pancreatic version of which is discussed below). Somatostatin has been reported to regulate the physiological function of various sites including the pituitary, pancreas, intestine and brain. It inhibits the release of growth hormone, insulin, and glucagon. It has many biological roles, including: inhibition of basal and stimulated hormone secretion from endocrine and exocrine cells, locomotion (locom).
Effects on total activity and cognitive function, and possible therapeutic value in small cell lung cancer. Reubi, J .; C. Et al, Endocrinol
, og, 110, 1049 (1982). Conjugated somatostatin may be used to treat giant disease in children or acromegaly in adults.

(Thyrotropin-releasing hormone (THR) and analogs (SEQ ID NO: 202-
214)) THR stimulates the production of thyroid stimulating hormone (TSH, also known as thyrotropin) and prolactin secretion. In the adult, TSH is responsible for general upregulation of protein synthesis and induction of positive nitrogen equilibrium. In the embryo, it is required for normal development. Hypothyroidism in the embryo is the cause of cretin disease. Cretin disease has multiple congenital defects (congeni).
tal defect) and mental retardation. The conjugated THR can then be used as a therapeutic agent in the treatment of these disorders. It can also be used to treat thyroid dysfunction caused by the pituitary gland or in the diagnosis of thyroid tumors in humans.

C. Thyroid Hormone (Calcitonin (CT) and Calcitonin Precursor Peptide (SEQ ID NO: 215)
~ 224)) Calcitonin (CT) is secreted by C cells of the thyroid gland 32
It is a peptide of amino acids. Calcitonin is used therapeutically to reduce the symptoms of osteoporosis, but the details of its mechanism of action remain unclear. However, it was observed to induce parathyroid hormone (PTH) synthesis in cells from which CT was isolated, which leads to increased plasma CA ²⁺ levels in vivo. Furthermore, C
T has been shown to reduce the synthesis of osteoporin (Opn), a protein produced by osteoclasts and responsible for osteoclast attachment to bone. Therefore, the use of conjugated CT as a therapeutic peptide increases plasma Ca ²⁺ via PTH induction and reduces bone resorption by reducing osteoclast binding to bone. .

(Calcitonin gene-related peptide (CGRP) (SEQ ID NOs: 225 to 253)
) CGRP is a 37 amino acid peptide. This peptide results from alternative splicing of calcitonin gene transcripts. It exists in at least two forms: α-CGRP (or CGRP-I) and β-CGRP (or CGRP-II). CGRP shares considerable homology with amylin and adrenomedullin and is widely distributed in organs both centrally and peripherally. These organs include: skin, heart, pancreas, lungs and kidneys. CG
RP has many biological roles that affect the nervous and cardiovascular system, inflammation and metabolism.

D. Parathyroid Hormone and Related Peptides Parathyroid Hormone (PTH) (SEQ ID NOs: 254-293) Parathyroid Hormone (PTH) is an epithelial cell in response to systemic Ca ²⁺ levels. Synthesized and secreted by the parenchymal chief cells. It plays a major role in the regulation of serum calcium concentration, thereby affecting the physiology of mineral and bone metabolism. Parathyroid Ca ²⁺ receptors respond to Ca ²⁺ by increasing intracellular levels of PKC, Ca ²⁺ and IP ₃ ; after this step, PTH secretion follows the protein synthesis period. Although PTH synthesis and secretion in host cells is constitutive, Ca ²⁺
By increasing the rate of PTH proteolysis when a ²⁺ levels are elevated and by decreasing PTH proteolysis when Ca ²⁺ levels are reduced, the level of PTH in the host cells (and thus its secretion) is reduced. ). The role of PTH is to regulate Ca ²⁺ concentration in extracellular fluid. Thus, the feedback loop that regulates PTH secretion involves the parathyroid gland, Ca ²⁺ , and the target tissues described below.

PTH acts by binding to the cAMP-coupled plasma membrane receptor,
Initiate a cascade of reactions that peak in the biological response. PT of the body
The response to H is complex but is directed to increasing Ca ²⁺ levels in extracellular fluid in all tissues. PTH induces bone lysis by stimulating osteoclast activity, which leads to elevated plasma Ca ²⁺ and phosphate. In the kidney, PTH reduces renal Ca ²⁺ clearance by stimulating its reabsorption; at the same time, PTH reduces phosphate reabsorption,
And thereby increase the clearance. Finally, PTH is the liver,
It acts on the kidneys and intestines to stimulate the production of the steroid hormone 1,25-dihydroxycholecalciferol (calcitriol). This steroid hormone is responsible for Ca ²⁺ absorption in the intestine. Conjugated PTH may be used to regulate calcium homeostasis in patients with parathyroid hormone deficiency. Inhibitor analogs may be used to block PTH effects in renal failure patients or other patients with excess PTH levels.

Parathyroid hormone-related protein (PTHrP) (SEQ ID NOs: 294-309
)) Parathyroid hormone-related protein (PTHrP) has been addressed as a physiological regulator that reduces chondrocyte differentiation and prevents apoptotic cell death. PTHrP was first identified as a tumor-derived secreted protein with structural similarities to parathyroid hormone (PTH), a major regulator of calcium homeostasis. PTH and PTHrP bind to a common G protein-coupled cell surface receptor (PTH / PTHrP or PTH-1 receptor). This receptor is the N-terminal region of these peptides (1-34
) Is recognized. Thus, when tumor-derived PTHrP enters the circulation, it activates receptors in classical PTH target organs (eg bone and kidney) and induces PTH-like bioactivity. By promoting bone resorption and inhibiting calcium excretion, circulating PTHrP gives rise to the normal neoplastic paraneoplastic syndrome of humoral hypercalcemia associated with malignancy.

Initially found in tumors, PTHrP was subsequently shown to be expressed in a wide variety of normal tissues including the fetal and adult skeleton. When acting in concert with its amino-terminal PTH-1 receptor, PTHrP acts to regulate cell proliferation and differentiation. The anabolic effect of intermittent PTH administration on bone and its therapeutic potential in osteoporosis has been extensively investigated. PTHrP
Against the recognition that it is an endogenous ligand for the PTH / PTHrP receptor in osteoclasts, its use as an anabolic agent was also investigated. Modified PT
HrP peptides can be used for similar indications as PTH.

E. Pancreatic Hormone The major role of pancreatic hormones is in the body's overall energy metabolism by regulating the concentration and activity of many enzymes involved in catabolism and assimilation of the main cell energy supply. Adjustment.

Amylin (SEQ ID NOs: 310-335) Amylin, a pancreatic β-cell hormone, is a 37 amino acid peptide related to CGRP and calcitonin.
It is co-secreted with insulin from pancreatic β cells. Amylin is deficient in type 1 diabetes. Amylin appears to work with insulin to regulate plasma glucose levels in the bloodstream. This halts peptideprandial secretion after glucagon treatment and suppresses the rate of gastric emptying. People with diabetes have a deficiency in the secretion of amylin in parallel with a deficiency in insulin secretion, which results in an excessive influx of glucagon into the bloodstream during the post-treatment peptiderandial period.

Although insulin replacement therapy is the basis of diabetes treatment, the replacement of the function of both amylin and insulin may allow a more complete restoration of the normal physiology of glucose control. Type 2 diabetes is characterized by islet amyloid deposits. This islet amyloid deposit is composed primarily of the amyloidogenic human form of the islet amyloid polypeptide. Conjugated amylin may be used in the management of diabetes to limit postprandial hyperglycemia.

(Glucagon (SEQ ID NOS: 336 to 376)) Glucagon is a 29-amino acid hormone synthesized by α cells of the islets of Langerhans as a very large proglucagon molecule. Like insulin, glucagon lacks plasma carrier proteins, and like insulin, its circulating half-life is also about 5 minutes. As a result of the latter trait, the main effect of glucagon is on the liver, the first tissue into which blood containing pancreatic secretions is poured. Glucagon binds to the plasma membrane receptor and is coupled to adenylate cyclase via the G protein. The resulting increase in cAMP and PKA reverses all of the above effects that insulin has on the liver. This increase also leads to a marked increase in circulating glucose, which is derived from hepatic gluconeogenesis and hepatic glycogenolysis. The conjugated glucagon construct can be used to manage unstable diabetes with recurrent hyperglycemia or to prevent or treat iatrogenic hyperglycemia.

Insulin and Insulin-Like Peptides (SEQ ID NOS: 377-382) The first of these recognized hormones was insulin, a 21- and 30-amino acid disulfide-bonded dipeptide produced by the pancreas. The major function of insulin is to block the coordinated action of many hyperglycemic hormones and to maintain low blood glucose levels. Insulin is IGF
-1, IGF-2 and relaxin are members of a family of structurally and functionally similar molecules. Although the three-dimensional structure of all four molecules is similar and they all have growth-promoting activity, the predominant role of insulin is metabolism, while the predominant role of IGF and relaxin is that of cell proliferation and differentiation. In adjustment.

Insulin is synthesized as a preprohormone in islets of Langerhans b cells. The signal peptide is removed in the vesicles of the endoplasmic reticulum, and it is packaged into secretory vesicles in the Golgi, folded into its native structure, and locks its conformation by the formation of two disulfide bonds. Specific protease activity cleaves the central third of the molecule, dissociating as the C peptide, leaving the amino terminal B peptide disulfide attached to the carboxy terminal A peptide.

Insulin produces its intracellular effect by binding to the plasma membrane receptor, which is the same on all cells. This receptor is a disulfide-bonded glycoprotein. One function of insulin (apart from its role in signal transduction) is to increase glucose transport in extrahepatic tissues, by increasing the number of glucose transporter molecules in the plasma membrane. The glucose transporter is in continuous turnover. The increase in plasma membrane content of the transporter results from the increasing rate of recruitment of new transporters to the plasma membrane. It is derived from a special pool of preformed transporters localized in the cytoplasm.

In addition to its role in regulating glucose metabolism (and its therapeutic use in treating diabetes), insulin stimulates lipid biosynthesis, reduces lipolysis, and amino acids to cells. Increase transport. Insulin also regulates transcription and modifies the cellular content of many mRNAs. This is an effect that stimulates proliferation, DNA synthesis and cell replication and that insulin has in common with IGF and relaxin. Therefore, conjugated insulin can be used to manage diabetes.

Neuropeptide Y (SEQ ID NOS: 383-389) Neuropeptide Y (NPY), a peptide with 36 amino acid residues, is one of the most abundant neuropeptides in both the peripheral and central nervous systems. Is. Neuropeptides belong to the pancreatic polypeptide family of peptides. The related molecule, peptide YY (PY
Similar to Y) and pancreatic polypeptide (PP), NPY is bent into a hairpin configuration that is important in aligning the free ends of the molecule for binding to the receptor.

NPY plays a broad role in the central nervous system (CNS) and the periphery.
Its CNS effects include major effects on feeding and energy expenditure and changes in heart rate, blood pressure, alertness and mood. In the periphery, NPY causes vasoconstriction and hypertension; it is also found in the gastrointestinal and genitourinary tracts, affecting its function by acting on gastrointestinal and renal targets. In a recent study, hypothalamic NPY was found to play a fundamental role in developing the features of obesity, a key pathway in regulating body fat signaling to the hypothalamus and body fat content. It is a good transducer. Leptin, an obesity gene product, induces NPY in obese (ob / ob) mice.
It has been found to reduce gene expression. Insulin and corticosteroids also reduce insulin NPY expression and corticosteroids reduce N
It increases PY expression and is involved in the regulation of hypothalamic NPY synthesis. Conjugated NP
Y may be used to treat MODM (Type II diabetes) in obesity and obese patients.

(Pancreatic Polypeptide (PP) (Sequence 390 to 396)) Pancreatic Polypeptide (
PP) is a 36 amino acid hormone produced by F cells within the pancreatic islets and exocrine pancreas. It is a member of the PP fold family of regulatory peptides, which increases glycogenolysis and regulates gastrointestinal activity. Thus, conjugated pancreatic polypeptides can be used to modify food absorption and metabolism.

Peptide YY (SEQ ID NOS: 397-400) PYY is a 36 amino acid long peptide that was first isolated from porcine intestinal tissue and is primarily localized to enteroendocrine cells. It has many biological activities, including: a wide range of activities within the digestive system and potent inhibition of intestinal electrolytes and fluid secretion.

(Somatostatin (SEQ ID NOS: 171-201)) Somatostatin secreted by pancreatic d cells is the same as somatostatin secreted by the hypothalamus.
It is a peptide of amino acids. In neural tissue, somatostatin inhibits GH secretion and therefore has a systemic effect. In the pancreas, somatostatin acts as a paracrine inhibitor of other pancreatic hormones and therefore also has systemic effects. It is speculated that somatostatin secretion is primarily responsive to blood glucose levels and increases as blood glucose levels increase, thus leading to a downregulation of glucagon secretion. The conjugated somatostatin can then be used to help control diabetes.

(F. Digestive Hormone) (Cholecystokinin (CCK) and Related Peptides (SEQ ID NOS: 401 to 416)
)) CCK is a 33 amino acid polypeptide originally isolated from porcine small intestine. It stimulates gallbladder contraction and bile flow and increases secretion of digestive enzymes from the pancreas. It exists in multiple forms, including CCK-
4 and CCK-8, with the octapeptide representing the predominant species with the greatest activity. It belongs to the CCK / gastrin peptide family and is distributed centrally to the nervous system and peripherally to the gastrointestinal system. It has many biological roles, including the following: stimulation of pancreatic secretions, gall bladder contraction, and intestinal mobility of the GI pathway, and possible mediators of satiety and pain stimulation. . The conjugated CCK can be used in diagnostic studies of the gallbladder or in chronic cholecystitis.

(Gastrin-releasing peptide (GRP) (SEQ ID NOS: 417 to 429)) GRP
Is a 27 amino acid peptide originally isolated from porcine non-stomach tissue,
And it is a homologue of the frog skin peptide named bombesin proliferation. It is widely distributed both centrally and peripherally in tissues including the brain, lungs and gastrointestinal tract. It regulates the physiological processes of various cells. These processes include secretion, smooth muscle contraction, neurotransmission and cell proliferation. Conjugated GRP can be used in the treatment of paralytic ileus or constipation in the elderly.

Gastrin and related peptides (SEQ ID NOS: 417-429) -Gastrin stimulates acid and pepsin secretion produced by the antrum of the stomach, 17
It is a polypeptide of amino acids. Gastrin also stimulates pancreatic secretions. Multiple active products are produced from gastrin precursors, and there are multiple control points in gastrin biosynthesis. Biosynthetic precursors and intermediates (progastrin and Gl
y-gastrin) is a putative growth factor; their products, amidated gastrin, are epithelial cell proliferation, differentiation of acid-producing mural cells and histamine-secreting enterochromaffin-like (ECL) cells, and in ECL cells. It regulates expression of genes associated with histamine synthesis and storage, as well as acute stimulatory acid secretion. Gastrin also stimulates the production of members of the epidermal growth factor (EGF) family, which in turn inhibits parietal cell function but stimulates proliferation of surface epithelial cells. Plasma gastrin levels are elevated in subjects with Helicobacter pylori, which subjects are known to have an increased risk of duodenal ulcer disease and gastric cancer. Therefore, the use of gastrin or gastrin antagonists as therapeutic agents can contribute to treat major upper gastrointestinal tract diseases.

Gastrin Inhibitory Peptides (SEQ ID NOS: 417-429) -Gastrin inhibitory peptides are 43 amino acid polypeptides that inhibit the secretion of gastrin. Conjugated GIP can be used to treat severe peptic ulcer disease.

Motilin (SEQ ID NOs: 430-433) -Motilin regulates gastrointestinal muscles,
It is a 22 amino acid polypeptide. Motilin-producing cells are distributed in duodenum, upper jejunum and colorectal adenocarcinoma and midgut carcinoma. Motilin stimulates intestinal motility.

Secretin (SEQ ID NOs: 434-441) -Secretin is secreted by the duodenum at pH values below 4.5 and stimulates pancreatic acinar cells to bicarbonate and H _2.
It is a 27 amino acid polypeptide that releases O. Secretin is a neurotransmitter (chemical messenger) in the neuropeptide family. Selectin is one of the hormones that control digestion (gastrin and cholecystokinin are the other hormones). Selectin is a polypeptide composed of 27 amino acids and is secreted by cells in the digestive system when the stomach is empty. Secretin helps the pancreas produce digestive juices rich in bicarbonate, which neutralizes intestinal acidity, the stomach produces pepsin, an enzyme that helps digest proteins, and the liver produces bile. stimulate.

Secretin may be useful in treating autism. In one study, children with disorders in the autism range received upper gastrointestinal endoscopy and intravenous administration of secretin to stimulate pancreatic bile secretion. All three had an increased pancreatic choleretic response when compared to non-autistic patients (7.5-10 m vs 1-2 mL / min).
L / min). Within 5 weeks of secretin infusion, a significant improvement in pediatric gastrointestinal symptoms was observed, as was a dramatic improvement in its behavior, which was manifested by improved eye contact, vigilance and expansion of expressive language. . These clinical observations
We suggest an association between gastrointestinal and brain function in patients with autistic behavior.

Vasoactive intestinal polypeptide (VIP) and related peptides (SEQ ID NO: 44)
2-464) -VIP is a 28-residue polypeptide produced by the hypothalamus and GI tract. VIP is to relax GI, inhibits acid and pepsin secretion, acts as a neurotransmitter in peripheral autonomic nervous system, and increases secretion of H ₂ O and electrolytes from pancreas and gut. VIP was originally found in the lung and intestine. And VIP has also been found in tissues including brain, liver, pancreas, smooth muscle and lymphocytes. VIP is structurally related to a family of peptides including PACAP, PHI, secretin and glucagon.
VIP has a diverse spectrum of biological actions, including vasodilation, electrolyte secretion, regulation of immune function and neurotransmission. The conjugated VIP may be useful in treating hydrochloric acid deficiency, ischemic colitis and irritable bowel syndrome (IBS).

G. The natriuretic peptide-natriuretic peptide hormone family includes three members involved in the regulation of blood pressure and fluid homeostasis: atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP,
Brain natriuretic peptide) and C-type natriuretic peptide (CNP)) are present.

Atrial Natriuretic Peptide (ANP) (SEQ ID NOs: 465-507) -AN
P is a 28 amino acid peptide hormone containing a disulfide bond. AN
P exerts natriuretic, diuretic and vasorelaxant effects and plays an important role in the body's blood volume and blood pressure homeostasis. Sm
ith, F.I. G. Et al., J. Dev. Physiol. 12, 55 (1989). The mechanisms that control ANP release have been the subject of intense investigation and are now fairly well understood. The major determinant of ANP secretion is myocyte elongation. Much less is known about the factors that regulate BNP release from the heart, but myocyte elongation has also been reported to stimulate BNP release from both the atria and ventricles. However, it has not been demonstrated that wall elongation acts directly or via factors released in response to dilation, such as endothelin-1, nitric oxide or angiotensin II. Recent studies indicate that by stimulating the A-type receptor for endothelin, endothelin plays an important physiological role as a mediator of acute dose-load-induced ANP secretion from atrial myocytes in rational animals. In fact, endogenous paracrine / autocrine factors released in response to atrial wall elongation, but not direct elongation, are almost exclusively found for ANP secretion during combined inhibition of endothelin A / B and angiotensin II receptors. A as a function of capacity load, as evidenced by complete interruption
It seems to be responsible for the activation of NP secretion. Furthermore, under certain experimental conditions, angiotensin II and nitric oxide may also exert a significant regulatory effect on elongation-activated ANP secretion. The molecular mechanism by which endothelin-1, angiotensin II and nitric oxide synergistically regulate elongation-activated ANP release remains unclear.
Vol. 75, p. 876-885, published November 12, 1997, Jour.
nal of Molecular Medicine. Conjugated ANP is useful in the management of malignant hypertension or severe hypertension and renal failure.

Brain Natriuretic Peptide (BNP) (SEQ ID NOs: 507-516) -Brain Natriuretic Peptide (BNP) is a member of the natriuretic peptide family and is produced and released from the ventricles. BNP regulates fluid volume, blood pressure and vascular tone through the type A guanylyl cyclase coupled receptor. BNPs such as atrial natriuretic peptide (ANP) play a role in electrolyte-fluid homeostasis. The conjugated BNP may be useful in managing heart failure.

C-type natriuretic peptide (CNP) (SEQ ID NOS: 517-524) -C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family.
, Is produced in vascular endothelial cells (EC) and acts as an endothelium-derived relaxing peptide. It is well known that atrial natriuretic peptide and brain natriuretic peptide are involved in the regulation of cardiovascular and endocrine functions as circulating hormones, but the role of C-type natriuretic peptide (CNP) remains unknown. .

CNP is found primarily in the central nervous system and vascular endothelial cells, while ANP and BNP are cardiac hormones. ANP is primarily synthesized in the atria of the normal adult heart, while BNP is produced by both the atria and ventricles.

H. Tachykinins (SEQ ID NOs: 525-627) -a common C-terminal sequence (F) required for full biological activity, including neurokinins A, B and substance P.
-X-GLM-NH ₂₎ sharing the family including neurokinin A and substance P peptide.

Neurokinin A-Neurokinin A is a decapeptide previously known as substance K. Neurokinin A is a member of the tachykinin family of neuropeptides, including substance P and neurokinin B. Neurokinin A exhibits a variety of activities associated with smooth muscle contraction, pain transmission, bronchoconstriction, vasodilation and regulation of the immune system.

Neuromedin-Neuromedin is a smooth muscle stimulating peptide and is usually divided into four groups: bombesin-like, cassinin-like, neurotensin-like and neuromedin U. These neuropeptides and their receptors are localized to all components of the HPA hypophysial pituitary axis, the only exception seems to be neurokinin B, which is found in the gland pituitary gland. Not detected. Neuromedins exert a wide variety of effects on the HPA axis, and their action on the adrenal gland suggests their involvement in the regulation of adrenal cortex proliferation, structure and function. Neuromedin can exert both direct and indirect effects on the adrenal cortex. Direct effects are demonstrated by stimulation of mineralocorticoid and glucocorticoid therapeutic peptides by isolated or cultured adrenocortical cells, and intracellular [Ca2 +] mobilization. On the other hand, indirect effects may be mediated by ACTH, arginine-vasopressin, angiotensin II, catecholamines, or by other modulators of medullary origin.

Substance P and Related Peptides-Substance P is an 11 amino acid peptide originally isolated from brain and intestine. Substance P has been proposed as a neuromodulator involved in pain transmission in the spinal cord. Substance P also affects smooth muscle contraction, lowering blood pressure, stimulating secretory tissue and releasing histamine from mast cells.

I. Renin-related peptides Angiotensin (SEQ ID NOs: 628-677) -Angiotensin is a 10 amino acid peptide derived from the enzymatic cleavage of a2-globin by the renal enzyme renin. The two C-terminal amino acids are then released to give angiotensin I,
It is responsible for essential hypertension through the stimulated synthesis and release of aldosterone from adrenal cells. It is a multifunctional hormone that regulates blood pressure, plasma volume, functional thirst for neurons, and water uptake.

Angiotensin II is an octapeptide derived from angiotensin I by angiotensin converting enzyme and is widely distributed both centrally and peripherally in organs such as heart, kidney and liver. Angiotensin IV is a terminal hexapeptide fragment of angiotensin II that is metabolically formed from either angiotensin I or angiotensin II by proteolytic cleavage. Angiotensin IV is a vascular control, cardiac growth,
Plays a role in renal blood flow and memory function.

Angiotensin II is a key peptide hormone that regulates vascular smooth muscle tone, blood pressure, free water uptake and sodium retention. Angiotensin II controls vascular homeostasis, which guarantees loss of intravascular volume by stimulating increased vasospasm tone, increased sodium retention and increased free water uptake.

Renin Substrates and Inhibitors (SEQ ID NOS: 678-684) -Renin is a highly specific aspartic protease, which is a differentiated smooth muscle in the vasculature of the kidney called granular epithelial cells. Synthesized and released by cells. Renin is specific for its substrate, angiotensinogen, and renin specifically cleaves angiotensinogen at the Leu ¹⁰ -Val ¹¹ bond,
It forms the decapeptide angiotensin I (AI). The renin-angiotensin system is involved in the control of fluid and mineral balance throughout vertebrates.
Renin is used in mammals, birds, reptiles, amphibians, teleosts, cartilaginous fish and jawless (a
Gnathan). Specific renin inhibitors also include, for example:
It may be designed for therapeutic applications for the treatment of hypertension and congestive heart failure (Bl
undell et al., 1987).

J. Endothelin and related peptides (SEQ ID NOs: 685-744) -The endothelin peptide family consists of the 21 amino acid isoforms endothelin-1, endothelin-2, endothelin-3, sarafotoxin (snake venom) and scorpion toxin.

Endothelin (ET) and large endothelin-endothelin are found in endothelial cells in a wide variety of organ systems. Examples of pathological and physiological processes associated with altered endothelin levels and synthesis include:
Effects on atherosclerosis and hypertension, coronary vasospasm, acute renal failure, changes in intracellular Ca2 ⁺ levels, and the renin-angiotensin system. Endothelin is released in response to variations in angiotensin II, vasopressin and cytokine (eg TGF-βm, TNF-α, IL-β-) levels, and other physiological events including increased blood flow.

The endothelin family of peptides consists of a very potent endogenous vasoconstrictor originally isolated from endothelial cell supernatants. They regulate blood flow to organs by delivering a vasoconstrictor effect to arteries. Endothelin is derived from large endothelin, which is transformed by a unique membrane-bound metalloproteinase endothelin-converting enzyme into a 21 amino acid bioactive form (ET-1,
ET-2 and ET-3).

Of the three isoforms (ET-1, ET-2, ET-3), endothelin-1 is the major isoform and plays an important role in regulating vascular function. Endogenous endothelin peptides and their receptors are found in blood vessels, uterus,
It is differentially distributed throughout many smooth muscle tissues, including the bladder and intestine. Through this widespread distribution and localization, they exert biological functions in regulating vascular tone and in causing mitogenesis. ETs and their receptor subtypes are also present in various endocrine organs. It appears to act as a modulator of secretion of prolactin, gonadotropin GH and TSH. Endothelin may also be a disease marker or etiological factor in ischemic heart disease, atherosclerosis, congestive heart failure, renal failure, systemic hypertension, pulmonary hypertension, cerebral vasospasm.

Exogenously administered endothelin-1 has been demonstrated to increase peripheral resistance and blood pressure in a dose-dependent manner. However, during the first few minutes of intravenous administration, endothelin also induces peripheral resistance and blood pressure, probably due to the release of vasodilator compounds such as nitric oxide, prostacyclin and atrial natriuretic peptide. Lower.

ET (A) Receptor Antagonists-Endothelin receptors exist in two forms: A (ET-A) and B (ET-B1 and ET-B2). E
The TA receptor is responsible, while ET-B1 and ET-B2 mediate vasorelaxation and vasoconstriction, respectively.

Sarafotoxin Peptide-As previously described, the endothelin (ET) peptide is a potent growth factor that binds to G protein coupled receptors. Atra
Sarafotoxin isolated from Ctaspis engaddensis (S6
) Is highly homologous to endothelin. Sarafotoxin peptides have significant vasoconstrictor activity and are responsible for the loss of ischemic limbs after being bitten by a snake or scorpion. These are vasoconstrictors (vasopre in shock and sepsis).
It can be used therapeutically as a peptidase stabilizing peptide as a ssive agent.

K. Opioid peptides (SEQ ID NOs: 745-927) -Opioids are a large class of drugs that are used clinically as analgesics, which include plant-derived and synthetic alkaloids and peptides endogenously found in the mammalian brain. Including both. Although plant-derived alkaloids have been known and used for thousands of years, endogenous opioid peptides were only discovered in the mid-1970s.

As opioids, there are casomorphin peptides,
Included are demorphins, endorphins, enkephalins, deltrophins, dinorphines, and analogs and derivatives thereof.

Casomorphin Peptide-Casomorphin peptide is a novel opioid peptide derived from casein (β-casomorphin). β-casomorphine is
A more extensively studied opioid peptide derived from dietary protein (β-casein). These were originally isolated from bovine β-casein.
This same sequence occurs in sheep and buffalo β-casein.

Demorphin-Demorphin is Phylomed
It is a 7 amino acid peptide originally isolated from frog skin of Usa sauvagei. Demorphin is a ligand that binds with high affinity to the mu opioid receptor and has many biological roles, including analgesia, endocrine regulation, immune regulation, increased K ⁺ conductance and inhibition of action potentials. Have.

Dynorphin / new endorphin precursor-related peptide-dinorphin is
It is a class of endogenous opioids that exist in multiple forms in the central nervous system. Dinorphine is derived from the precursor prodinorphin (Proenkephalin B).
Dinorphine, also known as dinorphine A1-17, has the sequence Tyr-Gly-
^{Gly-Phe-Leu 5 -Arg-} Arg-Ile-Arg-Pro 10 -Ly
s-Leu-Lys-Trp- Asp 15 -Asn-Gln ( SEQ ID NO: 1) is a well known opioid which has the. Dyn A1-13 (SEQ ID NO: 2), Dyn A2
-13 (SEQ ID NO: 3), Dyn A1-12, Dyn A2-12 and Dyn
A2-17, as well as amide analogs (see, eg, Lee et al., US Pat.
62,941), N-terminal truncated dinorphin analogs (eg, N described in International Patent Application WO 96/06626 to Lee et al.).
Terminally truncated dynorphin analogs) as well as des-Tyr or des-Ty
Analogues of r-Gly (see also International Patent Application WO 93/25 also to Lee et al.
Many derivatives and analogs of dinorphin are known, including analogs disclosed in 217). Dynorphi is a very potent opioid and exhibits a selective affinity for the kappa receptor. Goldst
ein, A .; , Peptides, Structure and Functi
on, Proceedings of the 8th American P
eptides Symposium, Hruby, V. J. And Rich,
D. H. Eds., 409 (1983).

Endorphins-Endorphins are derived from the precursor protein lipotropin. Endorphins have been found to elicit several biological responses, such as analgesia, behavioral changes and growth hormone release. Akil, H .; Et al., Ann. Rev. Neurosci. , 7,223 (1
984).

Enkephalins and related peptides-Enkephalins and endorphins are neurohormones that block the transmission of pain impulses. The activity of neurons in both the central and peripheral nervous systems is affected by numerous neurohormones that act on cells very far from their site of release. Neurohormones can modify the ability of nerve cells to respond to synaptic neurotransmitters. Several small peptides have been recently discovered that have profound effects on the nervous system, such as enkephalins (eg Met-enkephalin and Leu-enkephalin) and endorphins (eg β-endorphin). These three contain a common tetrapeptide sequence (Tyr-Gly-Gly-Phe) that is essential for their function. Enkephalins and endorphins function as natural analgesics or opiates and reduce the pain response in the central nervous system. Akil, H .; Et al., Ann. Rev. Neur
osci. , 7, 223 (1984).

L. Thymic peptide (SEQ ID NOs: 928-934) -The thymus is believed to be responsible for the development and regulation of T cell immunity in both infants and adults. The thymus appears to exert its regulatory function through the secretion of various acellular hormone-like products called thymic peptides through its epithelial cells.

Thymic peptides have been reported to have many effects on T cells.
Several studies have reported that thymic peptides can assist the development of immature progenitor cells into their fully competent T cells. Thymic peptides appear to regulate the expression of various cytokines and monokine receptors on T cells,
It induces the secretion of IL-2, interferon α and interferon γ (a substance that fights disease) when the immune system is challenged. There are reports that the use of thymic hormones in children with chemotherapy-induced immunodeficiency resulted in an increase in circulating T cells, normalization of T cell subsets, and repair of delayed hypersensitivity reactions.

Thymopoietin-Thymopoietin is the largest known thymic hormone and consists of 49 amino acids.

Thymulin-Previously known as thymic serum factor, thymulin is the smallest of the chemically characterized thymic hormones and consists of 9 amino acids. Cymulin is a hormone responsible for stimulating the production of immune system T cells.

Thymopentin-Thymopentin is a therapeutic peptide 5 or Timun.
A small, synthetic thymic peptide drug, also known as ox. In the US,
Thymo Pentine is an Immunobiology Research Ins
Developed by titute as an AIDS treatment. Thymopentin has been studied in more detail than most other thymic peptide drugs. At least one study claimed a significant increase in T cells and a slight clinical improvement in patients who received thymopentin three times a week compared to untreated control participants. Patients who took the drug showed greater "immunological stability" and some clinical improvement compared to 14 untreated control participants.

Thymosin-thymosin is a mixture of 15 or more proteins. One of these proteins is 28 amino acid thymosin alpha-1. Thymosin has therapeutic applications in renal dialysis patients for the treatment of primary immune deficiencies and as a booster for influenza vaccines. Thymosin has also been tested for activity against chronic hepatitis B and hepatitis C, HIV infection and certain forms of cancer in ongoing clinical trials.

Thymic fluid factor (THF) -THF is a thymic peptide currently being tested as an anti-HIV treatment. In a preclinical study in rats with CMV-related immunosuppression, THF restored immune capacity through regulation of T cells. Furthermore, THF is
May have therapeutic applications in the treatment of herpes (in at least one study)
Causes a rapid regression of viral infections and an increase in T cell populations.

L. Other Peptides Adrenomedullin Peptide (AM) (SEQ ID NOS: 935-945) Adrenomedullin is a potent vasodilator peptide that exerts major effects on cardiovascular function. Its systemic administration causes a rapid and severe decrease in blood pressure and an increase in pulmonary blood flow. Another of its effects is bronchodilation, which causes drunk behavior (dri
nking behavior) and angiotensin-induced aldosterone secretion. The Journal of B
iological Chemistry, Volume 270, No. 43, 25344.
See pages 25347, 1995 and references cited therein.

Aratostatin Peptides (SEQ ID NOS: 946-949) Aratostatin is a 6-18 amino acid peptide synthesized by insects to control the production of juvenile hormones. Juvenile hormones in turn regulate functions including metamorphosis and oogenesis. The aratostatin family of neuropeptides inhibits in vitro production of juvenile hormones, which regulate developmental and reproductive aspects in the cockroach Diploptera punctata, but are sensitive to inactivation by peptidases in the blood lymph, intestine. And binds to internal tissues.

(Amyloid β Protein Fragment (Aβ Fragment) (SEQ ID NO: 95)
0-1010)) These are the major components of amyloid plaques that accumulate intracellularly and extracellularly in neuritic plaques in the Alzheimer's disease brain. Aβ is a 4.5 kD protein, approximately 40-42 amino acids long, which is derived from the C-terminus of amyloid precursor protein (APP). APP is a transmembrane glycoprotein that is cleaved within the Aβ protein in the normal processing pathway to produce A
It produces α-sAPP, which is a secreted form of PP. The formation of α-aAPP hinders the formation of Aβ. Aβ accumulates due to abnormal processing of APP, resulting in
It has been proposed to search for compounds that inhibit the activity of the enzyme responsible for Aβ production. For example, Wagner et al., Biotech. Report (1994 /
1995), pp. 106-107; and Selkoe (1993) TINS 1.
6: 403-409. Under certain conditions, the Aβ peptide first aggregates and then deposits as a folded β-sheet structure characteristic of amyloid fibrils. The β-amyloid (1-42) form aggregates at a significantly faster rate and to a greater extent than β-amyloid (1-40).

Antimicrobial Peptides (SEQ ID NOS: 1011-1047) Antimicrobial peptides are key components of the innate immune system of most multicellular organisms,
It is active against one or more microorganisms such as bacteria, fungi, protozoa, yeasts, and mycobacteria. Examples of such peptides include defensins, cecropins, buforins, and magainins. Despite wide branching in sequence and classification, most antimicrobial peptides share a common mechanism of action (ie, pathogen membrane permeabilization). Antimicrobial peptides are divided into two broad groups: linear and cyclic. In linear antimicrobial peptides, there are two subgroups: linear peptides that tend to adopt an α-helix amphipathic conformation and unusual composition rich in amino acids such as Pro, Arg, or Trp. Linear peptide of. The cyclic group includes all cysteine containing peptides,
And further, it can be divided into two subgroups corresponding to single or multiple disulfide structures.

Most antimicrobial peptides cause an increase in plasma membrane permeation. Inhibition of specific membrane proteins, synthesis of stress proteins, termination of DNA synthesis, cleavage of single-stranded DNA by defensins, interaction with DNA by buforin (without termination of synthesis), or production of hydrogen peroxide. However, there is also evidence of other mechanisms. Antimicrobial peptides may also act by inducing self-destructive mechanisms such as apoptosis in eukaryotic cells or autolysis in bacterial targets. Antimicrobial peptides are also known to act as inhibitors of enzymes produced by pathogenic organisms, either by acting as pseudosubstrates or by tightly binding to active sites that interfere with substrate access. Has been.

Increased levels of antimicrobial peptides have been demonstrated in some animal and human infections (
For example, Mycobacterium, Pasteurella, or Cry
α-defensins in ptoporidium infections), and various peptides have been reported in blisters and wound fluids. Inflammatory conditions or irritation are also associated with the induction of antibiotic peptides.

Depleted levels of antimicrobial peptides are associated with several pathologies. Thus, patients with specific granule deficiency syndrome are completely deficient in α-defensin and suffer from frequent and severe bacterial infections. Low levels of hista (HISTA) from saliva of HIV patients
tin) was associated with a high incidence of oral candidiasis and fungal infections. Perhaps the most compelling illustration of the relevance of antimicrobial peptides in human pathology comes from cystic fibrosis, a genetic disease associated with recurrent bacterial infections of the respiratory tract. The defective chlorine channels that cause this disease increase the salinity of the alveolar fluid, and thus
It impairs the antibacterial activity of β-defensins, which are sensitive to salt. Andreu D
． (Ed) (1998) "Antimicrobiological peptides" Bi
opolimers (Peptide Science) Vol. 47, No. 6, 4
Pages 13-491. A. Andreu, L .; Rivas (1998) Animal
Antimicrobial Peptides: An Overview,
Biopolymers (Pep. Sci.) 47: 415-433.

(Antioxidant Peptides (SEQ ID NOS: 1048 to 1050)) Antioxidants are agents that prevent oxidative damage to tissues. Mammalian cells are continuously exposed to reactive oxygen species such as superoxide, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. These reactive oxygen intermediates are used in aerobic metabolism, drug and other xenobiotic catabolism, ultraviolet and X-ray irradiation, and phagocyte (
For example, in response to a respiratory burst of white blood cells), it kills invading bacteria (eg, those introduced through a wound) that are produced by cells in vivo. For example, hydrogen peroxide can be generated by stressed and damaged cells, among others.
It is produced during the breathing of most living organisms.

One example of an antioxidant peptide is natural killer enhancing factor B (NKEF-B).
, Which belongs to the highly conserved family of recently discovered antioxidants. The natural killer enhancement factor (NKEF) has been identified and cloned based on its ability to increase NK cytotoxicity. Two genes (NKEF-
A and NKEF-B) encode the NKEF protein, and the sequence analysis shown suggests that each belongs to a highly conserved family of antioxidants. The role of NKEF-B as an antioxidant was demonstrated by its protection of transfected cells against oxidative damage by hydrogen peroxide. NKEF-
B has antioxidant activity towards pro-oxidants such as alkyl hydroperoxides and MeHg. Induction of NKEF-B by HP, along with its antioxidant activity, indicates that NKEF-B is an important oxidative stress protein that provides protection against various xenobiotic toxic agents.

Apoptosis-Associated Peptides (SEQ ID NOS: 1051-1075) Animal cells can self-destruct through an intrinsic program of cell death (Stell).
er, 1995). Apoptosis is a form of programmed cell death characterized by specific morphological and biochemical properties (Wyllie et al.,
1980). Morphologically, apoptosis is a series of structural changes in dying cells, plasma membrane blebbing (ie, blistering), cytoplasmic and nuclear condensation, and cell fragmentation into membrane apoptotic bodies. ) (Stell
er, 1995; Wyllie et al., 1980).

Biochemically, apoptosis is characterized by the degradation of chromatin, which is initially a large fragment of 50-300 kilobases, followed by 200.
It becomes a smaller fragment that is a monomer and multimer of the base (Ob
erhammer et al., 1993; Wyllie, 1980). Another biochemical indicator of apoptosis is the protein clusterin (TRPM-2 or SGP-2.
Also known as) (Pearse et al., 1992) and activation of the type II transglutaminase, which crosslinks the protein to the apoptotic body coat (Fesus et al. 1991)
. Apoptosis is a complex phenomenon of related morphological and biochemical processes that can vary by tissue and cell type (Zakeri et al., 1995).
).

Performance of apoptosis minimizes leakage of cellular components from dying cells (apoptosis causes cells to regress). For example, proteases can damage adjacent cells or stimulate inflammatory responses. This basic feature of apoptosis distinguishes apoptosis from necrosis. Necrosis, which usually results from trauma, causes the damaged cells to swell and lyse, releasing cytoplasmic substances that stimulate the inflammatory response (Steller, 1995; Wyllie et al., 19).
80).

Bladder Cell Peptide (BCP) (SEQ ID NOS: 1076-1080) Neuropeptidergic cyst cells of the marine mollusc Alysia californica are involved in the spawning behavior of animals. These neurosecretory cells synthesize the egg-laying hormone (ELH) precursor protein and produce multiple bioactive peptides (including ELH), some cyst cell peptides (BCP), and acidic peptides (AP). . The sac cells of the marine mollusc Alysia californica are well-characterized neuroendocrine cells that initiate spawning. During sexual maturation, these cells (cystic neurons) develop the ability to store hormones released during periods of nervous system stimulation. This hormone is important for the process of spawning and should therefore not be released before the animal is sexually mature. Alpha-cyst cell peptides belong to a small family of structurally related peptides that can induce cyst cell activity in vitro.

(Bombesin (SEQ ID NOS: 1081-1090)) Bombesin has a common C-terminal sequence Trp-Ala-X-Gly-His-Me.
It is a bioactive tetradecapeptide neuropeptide that belongs to a family of peptides that share t-NH2 and the N-terminal region. It has a regulatory role found in nerves and intestines of the brain that prevents gastric injury due to the release of endogenous gastrin. A mammalian homolog of bombesin is a gastrin-releasing peptide (GRP).
).

Bone Gla Protein Peptides (SEQ ID NOs: 1091-1097) Osteocalcin (bone Gla protein or BGP) is produced and secreted by osteoblasts in the process of bone formation. Like collagen, this protein is a component of bone matrix. Serum osteocalcin is elevated when the rate of bone formation is increased. Levels are high during puberty when bone growth is fastest. Levels are often also high in diseases with high bone turnover, such as hyperparathyroidism and hyperthyroidism. In postmenopausal osteoporosis, osteocalcin levels are sometimes increased, which reflects an increase in turnover to rapid bone resorption of secondary bone. Lower levels of osteocalcin appear to be present in senile osteoporosis, which develops in older subjects, reflecting a decrease in both bone turnover and bone formation rates. . Treatment regimens that increase bone formation also increase serum osteocalcin levels.

CART Peptides (SEQ ID NOS: 1098-1100) Cocaine and amphetamine regulated transcript peptides (CART) are recently discovered hypothalamic peptides with potent appetite suppressant activity. In the rat, the CART gene encodes a peptide of either 129 or 116 amino acid residues, whereas in humans only the short form exists.
The putative signal sequence occurs in the 102 or 89 residue prohormone 27
It is an amino acid residue. The C-terminus of CART consists of 48 amino acid residues and 3 disulfide bonds and is believed to constitute the biologically active part of this molecule.

In the central nervous system, CART is highly expressed in many hypothalamic nuclei, some of which are involved in the regulation of feeding behavior. CART mRNA is
Leptin-regulated and expressed CART is a potent inhibitor of feeding that even abrogates the feeding response induced by neuropeptide Y. Therefore, the putative CART receptor is a strong therapeutic target for antiobesity drugs. CART, a new anionic peptide Th
im L; Kristensen P; Larsen PJ; Wulff BS
, Int J Biochem Cell Biol, 30 (12): 1281.
-4 1998 Dec.

Cell Adhesion Peptide (SEQ ID NO: 1101) Cell adhesion peptides are directly involved in the cellular response to external stimuli. For example, during the inflammatory response, leukocytes must move away from the cytoplasmic compartment to the point of antigenic injury. The mechanism of this migration event is a complex interaction between soluble mediators and membrane-bound cell adhesion molecules. Soluble cell chemoattractants are produced in tissues damaged by a variety of resident cells, creating a chemical gradient outside the cytoplasmic compartment. The interaction of these factors with their receptors on leukocytes results in the directed migration of leukocytes towards increasing concentrations of chemotactic factors. At the same time, various adhesion peptides are bound to their initial rolling (rolli) on endothelial tissue.
ng) mediates up-regulation on leukocytes, which binds to specific ligands on activated endothelial tissue and ultimately results in migration between endothelial cells to that tissue. This step in the cascade of events is mediated by the interaction of specific cell surface proteins termed "cell adhesion molecules". This molecule is
For example, E-selectin (ELAM-1, endothelial leukocyte adhesion molecule-1), ICAM
-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1).

(Chemotaxis Peptide (SEQ ID NOs: 1102 to 1113)) The chemotactic peptide is a leukocyte (white cell) or a leukocyte (leukoc).
yte), and macrophages that stimulate the migration of tissues into tissues at the site of infection or injury, or alternatively, interfere with the migration of these same cells away from these sites.

Complement Inhibitors (SEQ ID NOS: 1114-1120) Inhibition of complement attack on xenotransplants can be achieved by the use of complement inhibitors. Rejection of transplanted organs can include both extremely rapid hyperacute rejection (HAR) phases and slower cell rejection phases. The xenograft HAR binds to the preformed, endothelium of the donor organ.
Initiated by the "native" antibody, which binds to the tissue endothelium of the donor and activates complement attack by the recipient's immune system. Complement activation is a liquid-phase protein (C3a, C5a) and membrane-bound protein (C3b and C5b-) with chemotactic, procoagulant, proinflammatory, adhesive, and cytolytic properties.
9, ie C5b, C6, C7, C8, and C9). Complement inhibitors block this process.

Cortistatin Peptides (SEQ ID NOS: 1121-1124) Cortisstatin acts by antagonizing the effects of acetylcholine on cortical excitability, with its mRNA accumulating during sleep deprivation. , Thereby bringing about the synchronization of slow waves in the brain. Cortistatin-14 (CST-14) includes somatostatin-14 (SRIF-14) and its 1
It shares 11 of the 4 residues, yet its effects on sleep physiology, locomotor behavior, and hippocampal function are quite different from those of somatostatin.

(Fibronectin fragment and fibrin-related peptide (SEQ ID NO: 1
125-1174)) Fibronectin is a large glycoprotein composed of three types of blocks of repeating homologous peptide sequences. Some of the homologous blocks are
It forms functional domains that are organized in a linear array on two nearly identical subunit arms. Each arm can be divided into functional domains, which are often referred to by one of the substances that bind to that region (eg, heparin-binding fragments, fibrin-binding fragments, and cell-binding fragments).
. In some cell types, the cell-binding domain of fibronectin, Arg-
The Gly-Asp (RGD) sequence interacts with a cell surface glycoprotein called lib / IIIa. Fibronectin is also a component of extracellular and basement membranes,
Viral envelope glycoprotein, various bacteria (staphylococcus)
i and streptococci), and parasites (eg Tr
ypanosoma cruzi and Leishmania species).

Fibronectin has several adhesive functions (eg cell-cell adhesion, cell-cell adhesion).
Basement membrane adhesion, and clot stabilization). In addition, fibronectin promotes embryogenesis, neural regeneration, fibroblast migration, macrophage function, and binding of pathogens (viruses, fungi, bacteria, and protozoa) to mammalian cells and extracellular matrix. Therefore, fibronectin is involved in the pathogenesis of infection from the onset of infection through the final stages of wound healing. Proctor, R.M. A. , Re
v. Infect. Dis. , 9, 317 (1987).

FMRF and Similar Peptides (SEQ ID NOS: 1175-1187) FMRF is a neuropeptide encoded by the FMRF amide gene, which has a common C-terminal FMRF amide but different N-terminal extensions. FMRF amide-related peptide (FaRP) is ubiquitous in the animal kingdom and affects both neural and gastrointestinal function. Organisms have several genes encoding multiple FaRPs that have a common C-terminal structure but different N-terminal amino acid extensions.

Galanin and Related Peptides (SEQ ID NOS: 1188-1208) Galanin is a 29-30 amino acid peptide originally isolated from porcine small intestine. It has been found in two biologically active forms: GAL (1
~ 19) and the non-amidated form of GAL (1-30). Galanin has many biological roles, including: inhibition of release of biogenic amines in the hypothalamus, inhibition of presynaptic and postsynaptic cholinergic function, maintenance of gastrointestinal homeostasis. , And regulation of insulin and glucagon secretion.

Growth Factors and Related Peptides (SEQ ID NOS: 1209-1240) Growth factors are a family of proteins that regulate cell division. Some growth factors are cell type specific and stimulate division of only cells with the appropriate receptor. Other growth factors are more common in their effect. There are also extracellular factors that retard or interfere with division by antagonizing the effects of growth factors (eg,
Transforming growth factor β and tumor necrosis factor). These extracellular signals pass through cell surface receptors that are very similar to hormone receptors and through similar mechanisms (intracellular second messenger production, protein phosphorylation,
And, ultimately, alteration of gene expression).

(G Therapeutic Peptide Binding Protein Fragments (SEQ ID NOs: 1241-124
6)) Members of the G therapeutic peptide-binding regulatory protein (G protein) family transduce signals from membrane-bound receptors to intracellular effectors. This family includes G _s and G _{i, which} are responsible for the stimulation and inhibition of adenylate cyclase, respectively. Transducin (T) is localized to the disc membrane of the outer segment of the retinal rod and links photoactivation of rhodopsin to increased cyclic GMP phosphodiesterase activity. Originally found in bovine brain, G _o is the fourth member of this family.

Purified G-protein has similar physical properties. These are heterodimers composed of α subunit, β subunit, and γ subunit. The α subunit binds to and hydrolyzes the G therapeutic peptide. S. M. Mumby et al., PNAS 83,265 (1986) and Le.
See hinger 764.

Guaniline and Uroguanylin (SEQ ID NOS: 1247-1249) Guaniline and uroguanylin are peptides isolated from intestinal mucosa and urine that regulate cyclic GMP production in intestinal cells and induce guanylate. It binds to and activates cyclase C and regulates salt and water transport in many vertebrate epithelia, mimicking the action of some thermostable bacterial enterotoxins. In the kidney, both of these have well documented natriuretic and potassium excretory effects.

Chlorine secretion in the intestine is regulated by these hormones through activation of guanylate cyclase C (GC-C). Both peptides are expressed in various tissues and organs, including the kidney. Isolated, perfused kidneys and these hormones in vivo induce natriuresis and diuresis, but the localization and cellular mechanisms of their action in the kidney are still unknown.

(Inhibin peptide (SEQ ID NOS: 1250 to 1255) Inhibin has two subunits (α is 134 amino acids; β is 115 to 1).
16 amino acids). Its role is the inhibition of FSH secretion. The two inhibin isoforms (inhibin A and inhibin B) are produced by the gonads during gamete maturation and have different secretion patterns during the menstrual cycle. Inhibin is also produced by the placenta and embryonic membrane and may be involved in the physiological adaptation of pregnancy. Clinically, inhibin is a sensitive tumor marker in postmenopausal women or ovarian suppression (ovari) in infertile women.
They can serve as useful tools for assessing an reserve; they are also used in the diagnosis of maternal fetal disorders and can interfere with egg maturation or inhibit ovulation.

(Interleukin (IL) and interleukin receptor protein (
SEQ ID NOs: 1256-1263)) Interleukins are growth factors that target cells of hematopoietic origin. Various biological activities associated with immune and inflammatory responses have been attributed to interleukins. These responses include fever, cartilage destruction, bone resorption, thymocyte proliferation, activation of T and B lymphocytes, induction of acute phase protein synthesis from liver cells, fibroblast proliferation, and bone marrow cell proliferation. Differentiation and proliferation are included.

Laminin Fragments (SEQ ID NOS: 1264-1284) Laminin, a major non-collagen glycoprotein of the basement membrane, has been shown to promote adhesion, spreading, and migration of various tumor cell types in vitro. In particular, current major research in the laboratory utilizes intact laminin, purified proteolytic fragments of laminin, and synthetic peptides of laminin to identify functionally active sites in this large protein. Such basement membrane components are important modulators of proliferation, development and differentiation for various cell types. Conjugated laminin can be used to prevent tissue inflammation or fibrosis.

This category also includes the peptide Kringle-5 (ie K-5).
As used herein, the term "kringle 5" has three disulfide bonds that contribute to a particular three-dimensional conformation defined by the fifth kringle region of the mammalian plasminogen molecule. It refers to the area of mammalian plasminogen. One such disulfide bond links the cysteine residues located at amino acids 462 and 541 and the second bond is at amino acid 4
The cysteine residues located at positions 83 and 524 are linked, and the third bond is linked to the cysteine residues located at amino acids 512 and 536. The term "kringle 5 peptide peptide" refers to a peptide having a substantial sequence homologous to the corresponding peptide fragment of mammalian plasminogen and having anti-angiogenic activity between 4 and 104 amino acids (inclusive). Say.

(Leptin fragment peptide (SEQ ID NOS: 1285-12)
88)) Leptin, a protein product of the obesity gene, is secreted by adipocytes.
Leptin is involved in the regulation of body weight and metabolism in humans and may be involved in the pathophysiology of insulin resistance syndrome, which is associated with the development of cardiovascular disease.

Leucokinins (SEQ ID NOs: 1289-98) Leucokinins are a broad group of insect hormones that stimulate intestinal motility and tubular fluid secretion rate. In tubules, their primary action is to increase chloride permeability by binding to receptors on the basolateral membrane.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) (SEQ ID NOS: 1299-1311) This is a 38 amino acid peptide, which was first isolated from the sheep hypothalamus, which is also PACAP. It occurs in a 27 amino acid form called -27. PACAP is localized in the hypothalamus and elsewhere in the brain, respiratory tract and gastrointestinal system, which is involved in neurotransmission and hormonal function, energy metabolism. It has many biological effects, including the involvement of regulation of and neuroprotective activity.

Pancreastatin (SEQ ID NOS: 1312-1324) Pancreastatin is a 49 amino acid peptide that was first isolated, purified and characterized from porcine pancreas. Its biological activity in different tissues can be assigned to the C-terminal part of the molecule. Pancreatatin has a prohormone precursor, chromogranin A, which is a glycoprotein present in neuroendocrine, including the endocrine pancreas.

Polypeptides (SEQ ID NOS: 1325-1326) These are repetitive chains. Two examples are provided: (pro-Hyp-Gl
y) 10 ^* 20H20 and poly L-lysine hydrochloride.

Signal Transduction Reagents (SEQ ID NOS: 1273-167) Signal transduction is an extracellular signal (eg, chemical, mechanical, or electrical).
Are amplified and converted into a cellular response. Many reagents are involved in this process, including, for example, achatin-1, glycogen synthase, autocamtide 2, calcineurin autoinhibitory peptide, calmodulin-dependent protein kinase II, calmodulin-dependent protein kinase substrate, Calmodulin-dependent protein kinase substrate analog. CSK-17, Cys-Kemptide, Autocamtide 2
, Malantide, melittin, phosphate acceptor peptide, protein kinase C fragment, P34 cd2 kinase fragment, P
60c-src substrate II, protein kinase A fragment, tyrosine protein kinase substrate, syntide2, S6 kinase substrate peptide 32, tyrosine specific protein kinase inhibitor, and derivatives and fragments thereof.

Thrombin Inhibitors (SEQ ID NOS: 1368-1377) Thrombin is a key regulatory enzyme in the coagulation cascade; thrombin plays a dual role as both a positive and a negative feedback regulator. Be useful. In addition to its direct effect on hemostasis, thrombin
It has direct effects on various cell types that support and amplify the pathogenesis of arterial thrombosis. This enzyme is a potent activator of platelets, which aggregates platelets and releases substances such as ADP TXA.sub.2NE, which further increases the thrombotic cycle. Platelets within the fibrin mesh contain the main framework of the white thrombus. Thrombin also exerts a direct effect on endothelial cells, causing the release of vasoconstrictors and the translocation of adhesion molecules, which are the site of adhesion of immune cells. In addition, this enzyme causes mitogenesis of smooth muscle cells and proliferation of fibroblasts. From this analysis, it is clear that inhibition of thrombin activity by thrombin inhibitors constitutes a viable therapeutic approach towards reducing the proliferative events associated with thrombosis.

Toxins (SEQ ID NOS: 1387-1415) Toxins can be conjugated to target cancer cells, receptors, viruses, or blood cells using the present invention. Once the toxin binds to the target cells, the toxin is internalized, causing cytotoxicity and consequent cell death. Toxins are widely used as cancer therapeutic agents.

One example of a class of toxins is the mast cell degranulation peptide, 2 isolated from bee venom.
A cationic 22-amino acid residue peptide having two disulfide bridges,
It causes mast cell degranulation and, at low concentrations, histamine release,
It has anti-inflammatory activity at high concentrations. This mast cell degranulation peptide has potent anti-inflammatory properties and is 100-fold more effective than hydrocortisone in reducing inflammation. Due to their unique immunological properties, MCD peptides may serve as useful tools in studying the secretory mechanism of inflammatory cells such as mast cells, basophilic cells, and leukocytes, with therapeutic potential. Lead to the design of compounds with. An example of a mast cell granulating peptide is mastoparan, which results from wasp venom. Mastparan degranulates mast cells at a concentration of 0.5 μg / ml and releases histamine from the cells at the same concentration. IY. Hirai et al., Chem. Pharm
． Bull. 27, 1942 (1979).

Other examples of such toxins are omega-agatoxin TK, agerenin (age)
lenin), apamin, calcicudine, calciciptine, charbdotin (charbdot)
oxine), chlorotoxin, conotoxin, endotoxin inhibitor, geraphtoxin,
Iberiotoxin, kariotoxin
oxin), mast cell granulating peptide, margatoxin
), Neurotoxin NSTX-3, PLTX-II, thylatoxin (scyllato)
xine), stichodactyla toxin
n), and derivatives and fragments thereof.

Trypsin Inhibitors (SEQ ID NOS: 1416-1418) Trypsin inhibitors function as inhibitors of trypsin as well as other serine proteases. It is useful for treating lung inflammation, pancreatitis, myocardial infarction, and cerebrovascular ischemia.

Virus-Associated Peptides (SEQ ID NOS: 1419-1529) Virus-associated peptides are virus-associated proteins such as viral receptors, virus inhibitors, and envelope proteins. Examples include, but are not limited to: human immunodeficiency virus (
HIV), RS virus (RSV), human parainfluenza virus (HPV
), Measles virus (MeV), and simian immunodeficiency virus (SIV) peptide inhibitors, fluorogenic human CMV protease substrate, HCV core protein, HCV NS4A protein, hepatitis B virus receptor binding fragment, hepatitis B virus Pre- S region, herpesvirus inhibitor 2,
HIV envelope protein fragment, HIV gag fragment, HI
V substrates, HIV-1 inhibitor peptides, peptides T, T21, V3 decapeptides, virus replication inhibitor peptides, and fragments and derivatives thereof.

These peptides can be administered therapeutically. For example, peptide T is HIV
A chain of 8 amino acids from the V2 region of -1 gp120. These amino acids are
It is similar to that of the outer envelope of HIV. These amino acids are
Peptide T, which has been studied as a treatment for related neurological and systemic symptoms
Can reduce symptoms such as fever, night sweats, weight loss, and fatigue. It has also been shown to disperse psoriatic lesions.

(Various Peptides (SEQ ID NOS: 1529 to 1617)) Adjuvant peptide analogs, α-mating factor, antiarrhythmic peptides, anorexia-inducing peptides, α-1 antitrypsin, bovine pineal anti-reproductive peptide, valcin ( b
ursin), C3 peptide P16, cadherin peptide, chromogranin A fragment, contraceptive tetrapeptide, conantokin G (conantokin G)
), Conantkin T, crustacean heart-acting peptide, C-telopeptide, cytochrome b588 peptide, decorcin, delicious peptide, δ
Sleep-inducing peptide, diazepam binding inhibitor fragment, nitric oxide synthase blocking peptide, OVA peptide, platelet calpain inhibitor (P1)
, Plasminogen activator inhibitor 1, rigin, schizophrenia-related peptide, sodium potassium A-treated peptidase inhibitor-1 (
Sodium Potassium Atherapeutic Peptid
ease inhibitor-1), speract, sperm activation peptide, systemin, thrombin receptor agonist (three peptides), tufucin, adipokinin hormone, uremic pentapeptide, cryoprotective peptide, tumor necrosis factor, leech [Des Asp10] Decorsin, L-Ornityltaurine Hydrochloride, p-Aminophenylacetyl Tufucin,

[0169]

[Chemical 1] Sea urchin sperm activating peptide, SHU-9119 MC3-R and MC4R antagonists, glaspimod (immunostimulant, useful for bacterial infection, fungal infection, immunodeficiency, immune disorder, leukopenia), HP-228 ( Useful for melanocortin, chemotherapy-induced vomiting, toxicity, pain, diabetes mellitus, inflammation, rheumatoid arthritis, obesity), α-2-plasmin inhibitor (plasmin inhibitor), APC tumor suppressor (tumor suppressor, neoplasm) (Useful against), early pregnancy factor (immunosuppressive factor), endozepine diazepam binding inhibitor (receptor peptide), gamma interferon (useful against leukemia), gland kallikrein n-1 (immunostimulator), placental ribonuclease inhibitor , Sarcolecine ( Arcolecin) binding protein, surfactant protein D, Wilms tumor suppressor, Wilms' tumor suppressor, GABAB 1
b receptor peptide, prion-related peptide (iPrP13), choline-binding protein fragment (bacterial-related peptide), telomerase inhibitor, cardiostatin peptide, endostatin (end)
ostatin) -inducing peptides (angiogenesis inhibitors), prion inhibiting peptides, N-methyl D-aspartate receptor antagonists, C-peptide analogs (useful for diabetic complications).

2. Modified Therapeutic Peptides The present invention relates to modified therapeutic peptides and their derivatives. The modified therapeutic peptides of the present invention include a reactive group capable of reacting with an available reactive functional group of a blood component to form a covalent bond. The invention also relates to such modifications, such combinations with blood components and methods for their use. These methods are
Increasing the effective therapeutic in vivo half-life of the modified therapeutic peptide.

A wide variety of active carboxyl groups, especially esters, can be used as reactive groups to form covalent bonds with functionalities on proteins, where the hydroxyl moiety modifies the therapeutic peptide. It is physiologically acceptable at the level required. Many different hydroxyl groups can be used in these linking agents, but the most common are N-hydroxysuccinimide (NHS), and N-
Hydroxy-sulfosuccinimide (sulfo-NHS).

Primary amines are important targets for NHS esters as illustrated in Scheme 1A below. The accessible α-amine present at the N-terminus of the protein reacts with the NHS ester. However, the α-amino group of the protein is
It may not be desirable or available for S coupling. 5
Although some amino acids have a nitrogen in their side chain, only the ε-amine of lysine reacts significantly with NHS esters. The amide bond is formed when the NHS ester reacts with a primary amine to release N-hydroxysuccinimide in a ligation reaction, as shown in Scheme 1A below.

[0173]

[Chemical 2] In a preferred embodiment of the invention, the protein functional group is a thiol group and the reactive group is a maleimide-containing group such as γ-maleimido-butyramide (GMBA) or MPA. The maleimide group is most selective for the sulfhydryl group of the peptide when the pH of the reaction mixture is maintained between 6.5 and 7.4 as shown in Scheme 1B below. At pH 7.0, the reaction rate of maleimide groups with sulfhydryls is 1000 times faster than the reaction with amines. A stable thioether bond is formed between the maleimide group and the sulfhydryl, which cannot be cleaved under physiological conditions.

[0174]

[Chemical 3] The therapeutic peptides and peptide derivatives of the present invention can be modified for specific and non-specific labeling of blood components.

A. Specific Labeling Preferably, the therapeutic peptides of the invention are designed to specifically react with the thiol groups of mobile blood proteins. Such a reaction is preferably modified using a maleimide linkage (eg, prepared from GMBS, MPA or other maleimide) to a thiol group of mobile blood proteins such as serum albumin or IgG for therapeutic applications. Established by covalent attachment of peptides.

Under certain conditions, specific labeling with maleimide was performed using NHS and su
It offers several advantages over non-specific labeling of mobile proteins with groups such as lfo-NHS. Thiol groups are less abundant in vivo than amino groups. Therefore, the maleimide derivative of the present invention covalently binds fewer proteins. For example, in albumin, the most abundant blood protein, there is only one thiol group. Therefore, therapeutic peptide maleimide-albumin conjugates tend to include therapeutic peptide to albumin in a molar ratio of about 1: 1. In addition to albumin, IgG molecules (class II) also have free thiols. Since IgG molecules and serum albumin make up the majority of soluble proteins in blood, they also make up the majority of free thiol groups in blood available for covalent attachment to maleimide-modified therapeutic peptides. .

Furthermore, even among free thiol-containing blood proteins, specific labeling with maleimides results from the preferential formation of the therapeutic peptide maleimide-albumin conjugate due to the unique characterization of albumin itself. Lead to. The single free thiol group of albumin is highly conserved among species and is located at amino acid residue 34 (Cys ³⁴ ). Recently, it has been shown that Cys ^{34 of} albumin is highly reactive compared to the free thiols of other free thiol-containing proteins. This is due, in part, to the very low pK value (5.5) of albumin for Cys ³⁴ . This is generally much lower than the typical pK value for cysteine residues (typically about 8). Due to this low pK, under normal physiological conditions Cys ³⁴ of albumin is the predominantly ionized form, which dramatically increases its reactivity. In addition to the low pK value of Cys ^34, another factor which enhances the reactivity of Cys ³⁴ is its location,
Its location is a gap close to the surface of one loop of the V region of albumin.
This position makes Cys ³⁴ just available for all types of ligands,
It is an important factor in the biological role of Cys ³⁴ as a free radical trap and free thiol scavenger. These properties make Cys ³⁴ highly reactive with the therapeutic peptide maleimide, and the acceleration of the reaction rate can be 1000-fold compared to the reaction rate of the reaction of the therapeutic peptide maleimide with other free thiol-containing proteins. .

Another advantage of the therapeutic peptide maleimide-albumin conjugate is the reproducibility associated with a 1: 1 peptide-to-albumin specific for Cys ³⁴ . Other techniques, such as glutaraldehyde, DCC, EDC and other chemical activations for free amines, lack this selectivity. For example, albumin is 5
It contains two lysine residues, 25-30 of which are located on the surface of albumin and are accessible for binding. Activation of these lysine residues, or modification of peptides that bind through these lysine residues, results in a heterogeneous population of conjugates. Even when a 1: 1 molar ratio of peptide to albumin is used, the product consists of multiple conjugate acids, some of which contain 0, 1, 2 or more peptides per albumin, respectively. Has a peptide randomly attached to any one of the 25-30 available lysine sites. When many combinations are possible, characterization of the properties of the extracted components and their respective batches becomes difficult, reproducibility between batches is nearly impossible, making such conjugates less desirable as therapeutic agents. . Moreover, although conjugation of albumin via lysine residues appears to have at least the advantage of delivering more of the therapeutic agent per albumin molecule, studies have shown that a 1: 1 ratio of therapeutic agent to albumin is preferred. Was shown. Stehle et al., "The Loading Rate
Determines Tumor Targeting Property
s of Methotrexate-Albumin Conjugates
in Rats ", Anti-Cancer Drugs, Volume 8, 677-.
685 (1997), which is hereby incorporated by reference in its entirety.
In, the authors reported that a 1: 1 ratio of the anticancer drug methotrexate to albumin bound via glutaraldehyde gave the most promising results. While these conjugates were taken up by tumor cells, those with 5: 1 to 20: 1 methotrexate molecules had altered HPLC profiles and were rapidly taken up by the liver in vivo. It was inferred that at these higher ratios, conformational changes to albumin diminish its effectiveness as a therapeutic carrier.

Through the controlled administration of maleimide-therapeutic peptides in vivo, the specific labeling of albumin and IgG in vivo can be controlled. In a typical administration, 80-90% of the administered maleimide-therapeutic peptide labels albumin and less than 5% labels IgG. Microlabelling of free thiols such as glutathione also occurs. Such specific labeling is preferred for in vivo applications. This is because it allows an accurate calculation of the estimated half-life of the administered drug.

In addition to providing controlled and specific in vivo labeling, maleimide-therapeutic peptides can provide ex vivo specific labeling of serum albumin and IgG. Such ex vivo labeling involves the addition of maleimide-therapeutic peptides to blood, serum or saline containing serum albumin and / or IgG. Once modified ex vivo with a maleimide-therapeutic peptide, blood, serum or saline solution can be readministered into the blood for in vivo treatment.

In contrast to NHS-peptides, maleimide-therapeutic peptides are generally very stable in the presence of aqueous solutions and in the presence of free amines. Protecting groups are generally not required to prevent the maleimide-therapeutic peptide from reacting with itself, as the maleimide-therapeutic peptide reacts only with free thiols. In addition, the increased stability of the peptides allows the use of additional purification steps such as HPLC to prepare highly purified products suitable for in vivo applications. Finally, the increased chemical stability provides a product with a longer shelf life. B. Non-Specific Labeling The therapeutic peptides of the present invention can also be modified for non-specific labeling of blood components. Coupling with amino groups can be commonly used, especially with the formation of amide bonds for non-specific labeling. To form such a bond, a wide variety of active carboxyl groups, especially esters, can be used as the chemically reactive group linked to the therapeutic peptide, where the hydroxyl moiety is at the level required. Physiologically acceptable. Although a number of different hydroxyl groups can be used in these binders, N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS) are most convenient.

Other binders that can be utilized are described in US Pat. No. 5,612,034, which is incorporated herein by reference.

The various sites to which the chemically reactive groups of non-specific therapeutic peptides may react in vivo include cells, particularly red blood cells (mature red blood cells) and platelets, and proteins (
Examples thereof include immunoglobulins (including IgG and IgM), serum albumin, ferritin, steroid binding protein, transferrin, thyroxine binding protein, α-2-macroglobulin and the like. Those receptors to which the derivatized therapeutic peptides react, which are not long-lived, are generally cleared from the human host within approximately 3 days. The proteins shown above (including cellular proteins) should be at least 3 based on blood levels, especially with regard to half-life.
It can remain in the bloodstream for days, and can persist for 5 days or more (generally no more than 60 days, more typically no more than 30 days).

For the most part, the reaction involves mobile components in the blood, in particular blood proteins and cells, more particularly blood proteins and mature red blood cells. By "mobility" is meant that the component is not in place for any extended period of time (typically no more than 5 minutes, more typically 1 minute), but some Blood components can be relatively quiescent for long periods of time. Initially, there is a relatively heterogeneous population of labeled proteins and cells. However, for the most part, within days after administration, the population will differ substantially from the original population, depending on the half-life of the labeled protein in the bloodstream. Thus, typically within about 3 days or more, IgG becomes the predominant labeled protein in the bloodstream.

Typically, by 5 days post-administration, IgG, serum albumin and mature erythrocytes will be at least about 60 mol%, and usually at least about 75 mol% of the binding components in the blood, and IgG, IgM (to a substantially lesser extent). ) And serum albumin are at least about 50 mol% of the acellular binding component, usually at least about 75 mo.
It will be 1%, more usually about 80 mol%.

Desired conjugates of non-specific therapeutic peptides to blood components can be prepared in vivo by administering the therapeutic peptide directly to the patient. The patient can be a human or other mammal. The administration can be in bolus form or can be introduced slowly and over an extended period of time, such as by metering the flow and injecting.

If desired, the subject conjugates can also be prepared ex vivo by combining blood with a modified therapeutic peptide of the invention, wherein the reactive functional group on the blood component and the modified therapeutic peptide are combined. Allowing covalent attachment and then returning or administering the conjugated blood to the host. In addition, the above is to first purify individual blood components or a limited number of components (eg red blood cells, immunoglobulins, serum albumin, etc.) and then chemically purify that component or components in ex vivo. It can be achieved by combining with a therapeutically reactive therapeutic peptide. The labeled blood or blood component can then be returned to the host to provide the therapeutically effective conjugate to the subject in vivo. The blood is also
It can be treated to prevent coagulation during ex vivo manipulation.

3. Synthesis of Therapeutic Peptides Used in the Present Invention Peptide fragments can be synthesized by standard methods of solid-phase peptide chemistry known to those of skill in the art. For example, peptide fragments may be applied to the Applied Biosystems synthesizer.
and Edward (Steward, J.).
． M. And Young, J. et al. D. , Solid Phase Peptid
e Synthesis, Second Edition. , Pierce Chemical Com
pany, Rockford, III. , (1984)) by solid phase chemistry techniques. Multiple fragments are then similarly joined together and synthesized to form larger fragments. These synthetic peptide fragments can also be made with amino acid substitutions at specific positions.

For a solid phase peptide synthesis, an overview of many techniques is given in J. M. Stewart and J.M. D. Young, Solid Phase Peptide Synt
hesis, W.C. H. Freeman Co. (San Francisco)
1963 and J. et al. Meienhofer, Hormonal Protei
ns and Peptides, Vol. 2, p. 46, Academic Pre
ss (New York), 1973. For traditional solution synthesis, see G. Schroder and K.S. Lupke, The Pepti
See des, Volume 1, Acoustic Press (New York). Generally, these methods involve the sequential addition of one or more amino acids or appropriately protected amino acids to grow a peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected amino acid or derivatized amino acid is then
Appropriately protected complimen, attached to an inert solid support or under suitable conditions to form an amide bond.
tary) is utilized in solution by adding the next amino acid in the sequence with the (amino or carboxyl) group. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added and so on.

After all the desired amino acids have been joined within the appropriate sequence, all remaining protecting groups (and any solid support) are removed sequentially or simultaneously to give the final poly Nucleotides are formed. A simple modification of this general procedure allows for the addition of more than one amino acid at a time to grow a chain (eg, a protected tripeptide into an appropriately protected dipeptide ( By deprotection by attaching chiral centers (under non-racemization conditions) followed by formation of the pentapeptide).

A particularly preferred method of preparing compounds of the present invention involves solid phase peptide synthesis wherein the amino acid α-N-terminus is protected by an acid or base sensitive group. Such protecting groups should have the property of being stable to the conditions of peptide bond formation, while facilitating without breaking the growing peptide chain or racemization of any chiral centers contained therein. It should also have the property of being removable. A suitable protecting group is 9-fluorenylmethyloxycarbonyl (Fmoc
), T-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cb)
z), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxy. Carbonyl and the like. The 9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of therapeutic peptide fragments. Other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (p) for side chain amino groups such as lysine and arginine.
mc), nitro group, p-toluenesulfonyl group, 4-methoxybenzene-sulfonyl group, Cbz group, Boc group, and adamantyloxycarbonyl group; for tyrosine, benzyl group, o-bromobenzyloxycarbonyl group, 2,6 −
A dichlorobenzyl group, an isopropyl group, a t-butyl (t-Bu) group, a cyclohexyl group, a cyclophenyl group and an acetyl (Ac) group;
t-butyl group, benzyl group and tetrahydropyranyl group; for histidine, trityl group, benzyl group, Cbz group, p-toluenesulfonyl group and 2,4-dinitrophenyl group; for tryptophan, formyl group. For aspartic acid and glutamic acid, a benzyl group and t-
A butyl group and cysteine are triphenylmethyl (trityl) groups.

In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction, and are insoluble in the medium used. A preferred solid support for the synthesis of α-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly (styrene-1% divinylbenzene). A preferred solid support for the α-C-terminal amide peptide is 4- (2 ′, 4′-dimethoxyphenyl-Fmo.
c-aminomethyl) phenoxyacetamidoethyl resin (Applied)
Biosystems (available from Foster City, Calif.). This α-C-terminal amino acid is 4-dimethylaminopyridine (DMAP)
, 1-hydroxybenzotriazole (HOBT), benzotriazole-1-
N, N′-dicyclohexylcarbodiimide (DCC) with or without yloxy-tris (dimethylamino) phosphonium-hexafluorophosphate (BOP) or bis (2-oxo-3-oxazolidinyl) phosphine chloride (BOPCl) , N, N′-diisopropylcarbodiimide (DIC
) Or O-benzotriazol-1-yl-N, N, N ', N',-tetramethyluronium-hexafluorofluorophosphate (HBTU) is attached to the resin, the attachment being such as dichloromethane or DMF. It is mediated in a solvent at a temperature between 10 ° C and 50 ° C for about 1 to 24 hours.

When the solid support is 4- (2 ′, 4′-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin, the Fmoc group is coupled to the α-C-terminal amino acid described above. Prior to cleavage with a secondary amine, preferably piperidine. Deprotected 4- (2 ', 4'-dimethoxyphenyl-F
The preferred method for coupling to the moc-aminomethyl) phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N, N in DMF.
, N ', N',-Tetramethyluronium-hexafluorophosphate (HB
TU, 1 eq) and 1-hydroxybenzotriazole (HOBT, 1 eq)
Is. Sequential coupling of protected amino acids can be performed in automated polypeptide synthesizers well known in the art. In a preferred embodiment, the α-N-terminal amino acid of the growing peptide chain is Fmoc protected. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is achieved by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in approximately 3-fold molar excess and preferably the coupling is carried out in DMF. The coupling agent is usually O-
Benzotriazol-1-yl-N, N, N ′, N ′,-tetramethyluronium-hexafluorofluorophosphate (HBTU, 1 eq) and 1-hydroxybenzotriazole (HOBT, 1 eq).

At the end of solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously or in a single operation. Removal and deprotection of polypeptides can be accomplished using a cleavage reagent containing thioanisole, water, ethanedithiol and trifluoroacetic acid.
This can be accomplished in a single operation by treating the resin bound polypeptide. When the α-C-terminus of the polypeptide is an alkylamide, the resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by, for example, a transesterification reaction with methanol followed by aminolysis or direct transamidation. The protected peptide can be purified at this point or used directly in the next step. Removal of the side chain protecting group is accomplished by the cleavage cocktail (coc
ktail). Fully deprotected peptides can be of any or all of the following types: ion exchange on weak base resins (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (eg,
Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethylcellulose; partition chromatography (eg Sephadex G-25, LH-20) or countercurrent partition; high performance liquid chromatography (HPLC) (especially octyl-. Alternatively, it is purified by a series of chromatographic steps utilizing reverse phase HPLC on an octadecylsilyl-silica bonded phase packing.

The molecular weight of these therapeutic peptides was determined by the Fast Atom Bombardm.
ent (FAB) Mass Spectroscopy.

The therapeutic peptides of the invention can be synthesized with N-terminal and C-terminal protecting groups for use as prodrugs.

(1) N-Terminal Protecting Group As discussed above, the term “N-protecting group” means protecting the α-N-terminal of an amino acid or peptide, or otherwise amino acid or Refers to those groups intended to protect the amino group of a peptide against unwanted reactants during synthetic procedures. Generally, the N-protecting group used is Greene, "Pr.
active Groups In Organic Synthesis
(John Wiley & Sons, New York (1981)), which is incorporated herein by reference. In addition, protecting groups can be used, for example, as prodrugs that are readily cleaved in vivo by enzymatic hydrolysis to release the biologically active parent. α-N
The protecting group is a lower alkanoyl group (eg, formyl group, acetyl group (“Ac”)
), Propionyl group, pivaloyl group, t-butylacetyl group, etc .; 2-chloroacetyl group, 2-bromoacetyl group, trifluoroacetyl group, trichloroacetyl group, phthalyl group, o-nitrophenoxyacetyl group, chlorobutyryl group, benzoyl. Groups, other acyl groups including 4-chlorobenzoyl group, 4-bromobenzoyl group, 4-nitrobenzoyl group and the like; sulfonyl groups such as benzenesulfonyl group, p-toluenesulfonyl group and the like; for example, benzyloxycarbonyl Group, p-chlorobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, 2-nitrobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, 3,4-dimethoxybenzyloxycarbo group Group, 3,5-dimethoxybenzyl oxycarbonyl group,
2,4-dimethoxybenzyloxycarbonyl group, 4-ethoxybenzyloxycarbonyl group, 2-nitro-4,5-dimethoxybenzyloxycarbonyl group,
3,4,5-Trimethoxybenzyloxycarbonyl group, 1- (p-biphenylyl) -1-methylethoxycarbonyl group, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group, benzhydryloxycarbonyl group Group, t-butyloxycarbonyl group, diisopropylmethoxyoxycarbonyl group, isopropyloxycarbonyl group, ethoxycarbonyl group, methoxycarbonyl group, allyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, phenoxycarbonyl group, 4- Nitrophenoxycarbonyl group, fluorenyl-9-
Carbamate-forming groups such as methoxycarbonyl group, cyclopentyloxycarbonyl group, adamantyloxycarbonyl group, cyclohexyloxycarbonyl group, phenylthiocarbonyl group; for example, benzyl group, triphenylmethyl group, benzyloxymethyl group, 9-fluorenylmethyl Oxycarbonyl group (Fmoc
) And the like as well as silyl groups such as, for example, trimethylsilyl and the like.

((2) Carboxy Protecting Group) As discussed above, the term “carboxy protecting group” refers to blocking a carboxylic acid functional group while performing a reaction involving another functional group site of the compound. It refers to a carboxylic acid-protected ester group or amide group used for protection. Carboxy protecting groups are described by Greene, "Protective Gro
ups in Organic Synthesis, pp. 152-186 (19)
81), which is incorporated herein by reference. further,
Carboxy protecting groups may be readily cleaved in vivo by, for example, enzymatic hydrolysis to release the biologically active parent. Such carboxy protecting groups are well known to those skilled in the art and are described in US Pat. Nos. 3,840,556 and 3,719.
, 667, it is used extensively in the protection of carboxyl groups in the field of penicillins and cephalosporins. These disclosures are incorporated herein by reference. Representative carboxy protecting groups are C ₁ -C ₈ lower alkyl groups such as methyl, ethyl or t-butyl groups; arylalkyl groups such as, for example, phenethyl or benzyl, as well as alkoxybenzyl. Groups or their substituted derivatives such as nitrobenzyl groups; eg arylalkenyl such as phenylethenyl group; aryl and its substituted derivatives such as eg 5-indanyl group; eg dimethylaminoethyl group etc. Dialkylaminoalkyl groups such as, for example, acetoxymethyl group, butyryloxymethyl group, valeryloxymethyl group, isobutyryloxymethyl group, isovaleryloxymethyl group, 1- (propionyloxy) -1-ethyl group , 1- (pivaloyloxy) -1-ethyl group, 1- Alkanoyloxyalkyl groups such as tyl-1- (propionyloxy) -1-ethyl group, pivaloyloxymethyl group, propionyloxymethyl group; for example, cyclopropylcarbonyloxymethyl group, cyclobutylcarbonyloxymethyl group, A cycloalkanoyloxyalkyl group such as a cyclopentylcarbonyloxymethyl group and a cyclohexylcarbonyloxymethyl group; for example, an aroyloxyalkyl group such as a benzoyloxymethyl group and a benzoyloxyethyl group;
For example, an arylalkylcarbonyloxyalkyl group such as a benzylcarbonyloxymethyl group and a 2-benzylcarbonyloxyethyl group; for example, a methoxycarbonylmethyl group, a cyclohexyloxycarbonylmethyl group, a 1-methoxycarbonyl-1-ethyl group and the like. Such an alkoxycarbonylalkyl group or a cycloalkyloxycarbonylalkyl group; for example, a methoxycarbonyloxymethyl group, a t-butyloxycarbonyloxymethyl group, a 1-ethoxycarbonyloxy-1-ethyl group, a 1-cyclohexyloxycarbonyloxy group. An alkoxycarbonyloxyalkyl group such as a -1-ethyl group or a cycloalkyloxycarbonylalkyl group; for example, a 2- (phenoxycarbonyloxy) ethyl group, An aryloxycarbonyloxyalkyl group such as a-(5-indanyloxycarbonyloxy) ethyl group; and an alkoxyalkyl such as a 2- (1-methoxy-2-methylpropane-2-oiloxy) ethyl group Carbonyloxyalkyl group; for example, arylalkyloxycarbonyloxyalkyl group such as 2- (benzyloxycarbonyloxy) ethyl group and the like; for example, 2- (3-phenylpropen-2-yloxycarbonyloxy) ethyl group and the like Such as arylalkenyloxycarbonyloxyalkyl group; for example, alkoxycarbonylaminoalkyl group such as t-butyloxycarbonylaminomethyl group, etc .; for example, alkylaminoalkyl group such as methylaminocarbonylaminomethyl group, etc. Arylsulfonylamino alkyl group; for example, alkanoylamino group such as acetylamino methyl group; for example, 4-
Heterocyclic carbonyloxyalkyl groups such as methylpiperazinylcarbonyloxymethyl group; eg, dialkylaminocarbonylalkyl groups such as dimethylaminocarbonylmethyl group, diethylaminocarbonylmethyl group, etc .; eg (5-t-butyl (5- (lower alkyl) -2-oxo-1,3-dioxolen-4-yl) alkyl groups such as 2-oxo-1,3-dioxolen-4-yl) methyl group; and, for example, (5-phenyl-2-oxo-1
, 3-Dioxolen-4-yl) methyl group and the like (5-phenyl-2-oxo-1,3-dioxolen-4-yl) alkyl group.

Representative amidocarboxy protecting groups are aminocarbonyl groups and lower alkylaminocarbonyl groups.

Preferred carboxy protected compounds of the present invention include multiple compounds in which the protected carboxy group is a lower alkyl ester, cycloalkyl ester or arylalkyl ester (eg, methyl ester, ethyl ester, propyl ester, isopropyl ester. Ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester, etc.) or alkanoyloxyalkyl ester, cycloalkanoyloxyalkyl ester, aroyloxyalkyl ester or arylalkyl. It is a carbonyloxyalkyl ester. A preferred amidocarboxy protecting group is a lower alkylaminocarbonyl group. For example, aspartic acid can be protected at the α-C-terminus by an acid labile group (eg, t-butyl group) and at the β-C-terminus by a hydrogenation labile group (eg, benzyl group). It can be protected and then selectively deprotected during the synthesis.

Alternatively, it is also possible to obtain fragments of the peptides, for example by fragmenting the naturally occurring amino acid sequence using proteolytic enzymes according to methods well known in the art. In addition, the desired fragment of this therapeutic peptide can be obtained through the use of recombinant DNA technology using methods well known in the art.

Modification of Therapeutic Peptides The manner in which the modified therapeutic peptides of the invention are produced will vary widely depending on the nature of the various elements, including the molecule. The synthetic procedures are chosen to be simple, to provide high yields, and to allow highly purified, stable products. Usually, the reactive group is made as the final step, for example with a carboxyl group, and esterification to form the active ester is the final step in the synthesis. Specific methods for the production of the modified therapeutic peptides of the invention are described below.

Generally, the modified therapeutic peptides of the present invention will be blind.
Alternatively, it can be made using structure activity relationship (SAR) driven substitution. SAR is
An analysis that defines the relationship between the structure of a molecule and its pharmacological activity against a range of compounds. Various studies relating to individual therapeutic peptides have shown how the activity of the peptides varies according to variations in chemical structure or properties. More specifically, the free peptide is first assayed for therapeutic activity. The peptide is then modified according to the invention with only the linking group either at the N-terminus, C-terminus or internally of the peptide. Linking groups include the reactive groups discussed above. The therapeutic activity of the modified peptide linking group is then assayed and based on the detected activity a decision is made regarding the site of modification. The peptide conjugate is then prepared and its therapeutic activity determined. If the therapeutic activity of the peptide after conjugation is not substantially reduced (ie, the therapeutic activity is reduced by less than 1/10), the stability of the peptide is indicated by its in vivo longevity. To measure. If stability is not improved to the desired level, the peptide is modified at alternative sites. This procedure is then repeated until the desired level of therapeutic activity and the desired stability are achieved.

More specifically, each therapeutic peptide selected to undergo derivatization with a linker and a reactive group is modified according to the following criteria: The terminal carboxylic acid group is utilized in the therapeutic peptide. If possible, and the terminal carboxylic acid group is not important for retention of pharmacological activity, and no other sensitive functional groups are present in the therapeutic peptide, the carboxylic acid is modified for linker-reactive group modification. Is selected as the binding site of. If the terminal carboxylic acid group is involved in the pharmacological activity, or if the carboxylic acid is not available, then any other sensitive reactive group that is not important in retaining the pharmacological activity is linked to the linker-reactive Selected as a binding site for group modification. If some sensitive functional groups are available in the therapeutic peptide, after addition of a linker / reactive group and deprotection of all protected sensitive functional groups,
The combination of protecting groups is used in such a way that retention of pharmacological activity is still obtained. If sensitive functional groups are not available in the therapeutic peptide, or if a simpler modification pathway is desired, synthetic attempts will result in retention of biological activity and retention of receptor or target specificity. Allows modification of the native peptide in such a manner. In this case, the modification occurs at the opposite end of the peptide.

NHS derivatives can be synthesized from carboxylic acids in therapeutic peptides in the absence of other sensitive functional groups. In particular, such therapeutic peptides are reacted with N-hydroxysuccinimide in anhydrous CH ₂ Cl ₂ and EDC and the product is purified by chromatography or a suitable solvent to give the NHS derivative. Recrystallized from the system.

Alternatively, NHS derivatives can be synthesized from therapeutic peptides containing amino and / or thiol groups, and carboxylic acids. If free amino or thiol groups are present in the molecule, it is preferable to protect these sensitive functional groups before carrying out the addition of the NHS derivative. For example, if the molecule contains a free amino group, conversion of the amine to an Fmoc or preferably tBoc protected amine is required before carrying out the above chemistry. The amine functionality is not deprotected after preparation of the NHS derivative. Therefore, this method applies only to compounds in which the amine groups are not required to be free in order to induce the desired pharmacological effect. If the amino group is required to be free in order to retain the original biological properties of the molecule, another type of chemistry described in Examples 3-6 must be performed.

In addition, NHS derivatives can be synthesized from therapeutic peptides that contain amino or thiol groups and are carboxylic acid free. If the molecule of choice does not contain a carboxylic acid, a series of bifunctional linkers are used to treat the molecule with reactive NHS.
It can be converted into a derivative. For example, ethylene glycol bis (succinimidyl succinate) (E dissolved in DMF and added to free amino-containing molecules)
GS) and triethylamine (in a ratio of 10: 1, with EGS being preferred) produces the mono NHS derivative. N-[-maleimidobutyryloxy] succinimide ester (GMBS) and triethylamine in DMF can be used to generate NHS derivatives from thiol derivatized molecules. The maleimide group reacts with the free thiol and the NHS derivative is purified from the reaction mixture by chromatography on silica or by HPLC.

NHS derivatives can also be synthesized from therapeutic peptides containing multiple sensitive functional groups. In each case, the analysis and elucidation must be done in different ways.
However, thanks to the large amount of protecting groups and bifunctional linkers that are commercially available, the present invention preferably uses only one chemical step (described in Examples 1 or 3) to derivatize therapeutic peptides. As described above) or in 2 steps (including the pre-protection of sensitive groups described in Example 2) or 3 steps (protection, activation, and deprotection), applicable to any therapeutic peptide. is there. Only under exceptional circumstances,
In order to convert the therapeutic peptide into the active NHS or maleimide derivative, it is necessary to use multiple (more than 3) synthetic steps.

Maleimide derivatives can also be synthesized from therapeutic peptides containing free amino groups and free carboxylic acids. N- [γ-maleimidobutyryloxy] succinimide ester (GMBS) and triethylamine in DMF can be used to generate maleimide derivatives from amino derivatized molecules. The succinimide ester group reacts with the free amino and the maleimide derivative is reacted by crystallization, or by chromatography on silica, or by HPLC.
Purified from the reaction mixture.

Finally, maleimide derivatives can be synthesized from therapeutic peptides containing multiple other sensitive functional groups and no free carboxylic acids. If the molecule selected does not contain a carboxylic acid, a series of bifunctional cross-linking reagents are used to react the molecule with reactive NH.
It can be converted to the S derivative. For example, HBTU / HOBt / DIEA activation in DMF is used to couple maleimidopropionic acid (MPA) to a free amine to form a maleimide derivative through reaction of the free amine with the carboxylic acid group of MPA. obtain. Alternatively, a lysine residue can be added to the C-terminus of the peptide to allow conjugation at the amino group of lysine, as described in the Examples below. This added lysine allows for simple and efficient modification at the C-terminus of the peptide, while retaining the amide functional end capped, as designed by the initial choice of resin.

Many other commercially available heterobifunctional cross-linking reagents can alternatively be used when needed. A large number of bifunctional compounds are available for crosslinking to entities. Exemplary reagents include: azidobenzoyl hydrazide,
N- [4- (p-azidosalicylamino) butyl] -3 '-[2'-pyridyldithio) propionamide), bis-sulfosuccinimidyl suberate (sub)
erate), dimethyl adipimidate, disuccinimidyl tartrate, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-
Succinimidyl [4-azidophenyl] -1,3′-dithiopropionate,
N-succinimidyl [4-iodoacetyl] aminobenzoate, glutaraldehyde, and succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate.

[0212] Even more particularly, the peptide is preferably modified according to the nature of its substituents and the presence or absence of free cysteine. Most peptides can be grouped into three different classes: (1) cysteine-free peptides; (2) 1.
A peptide containing one cysteine, (3) a peptide containing two cysteines as a disulfide bridge (cysteine); and (4) a peptide containing multiple cysteines.

A. Cysteine-Free Peptides If the peptide does not contain cysteine, C-terminal additions are performed at all residues that are cleaved from the support resin and fully protected. EDC and NH
Liquid phase activation of the C-terminus with S can be reacted with the amino-alkyl-maleimide in one pot. The peptide is then completely deprotected. Alternatively, a lysine residue may be added to the C-terminus of the peptide, allowing modification at the epsilon amino group of lysine while maintaining the carboxy terminus capped with an amide group. Such addition of lysine residues is preferably performed only if the addition does not substantially affect the therapeutic activity of the peptide. The generalized reaction scheme for C-terminal modification of peptides that do not contain cysteine is illustrated in the schematic diagram below.

[0214]

[Chemical 4] N-terminal modifications are advantageous, and again, for peptides that do not contain cysteines, the addition at the N-terminus is carried out at all residues present on the support resin and is fully protected. Addition of an activated NHS-Mal bifunctional linker can be performed at the peptide present on the resin and at the deprotected N-terminus. The peptide is then completely deprotected. Examples of therapeutic peptides that are cysteine free and that undergo a C-terminal modification are described in Examples 7-26. Examples of therapeutic peptides that are cysteine free and undergo N-terminal modification are described in Examples 27-38. Generalized reaction schemes for N-terminal modification of cysteine-free peptides were described for hetero NHS maleimide (GMBS-like) and 3-MPA, respectively.
Is illustrated in the following schematic diagram.

[0215]

[Chemical 5]

[0216]

[Chemical 6] Alternatively, the peptide may be modified with internal amino acids (ie, neither C-terminal nor N-terminal). A generalized reaction scheme for modification at internal amino acids of peptides that do not contain free cysteine is illustrated in the schematic diagram below using homobis NHS and hetero NHS maleimide.

[0217]

[Chemical 7]

[0218]

[Chemical 8] Peptides that do not contain cysteine and can be modified as described above include: fragments of the Kringle 5 peptide, fragments of the GLP-1 peptide, fragments of dynorphin A, human growth hormone releasing factor, human neuropeptide Y. 1-24 fragment, and human secretin.
A full chemistry description of each of these peptides is reported in the Examples section.

B. Peptides Containing One Cysteine For peptides containing one cysteine, the cysteine must remain capped after addition of the maleimide. If cysteine is included in the binding site, an assessment should be made as to how much potency the cysteine was capped by the protecting group, resulting in a loss of potency. If the cysteine can remain capped, the synthetic route is similar to the route described in section A above for either the C-terminal modification or the N-terminal modification.

Alternatively, the peptide may be modified with internal amino acids (ie, neither C-terminal nor N-terminal). A generalized reaction scheme for modification of an internal amino acid of a cysteine-free peptide is illustrated in the schematic diagram below using homobismaleimide and hetero NHS maleimide (GMBS-like).

[0221]

[Chemical 9]

[0222]

[Chemical 10]   Examples of therapeutic peptides containing one cysteine include: G _α (Α subunit of G therapeutic peptide binding protein),
724-739 Fragment of nitric oxide synthase blocking peptide
G., human [Tyr0] inhibin α subunit 1-32 fragment, HI
254-274 fragment of the V envelope protein, and P34cdc
2 kinase fragment.

C. Peptide Containing Two Cysteines as a Disulfide Bridge (Cysteine) If the peptide contains two cysteines as a disulfide bridge, the peptide is cleaved from the support resin prior to the addition of maleimide. For modification of the peptide from the C-terminus, the carboxy terminus needs to be deprotected when the peptide is cleaved from the resin, which remains unprotected due to cleavage from the support resin. ) And two cysteines, all protecting groups are present. Mild air oxidation results in disulfide bridges, and the peptide can be purified at that stage. Liquid phase chemistry is then required to activate the C-terminus in the presence of the disulfide bridge and to add the maleimide to the C-terminus (via the amino-alkyl-maleimide). The peptide is then completely deprotected.

For modification of the peptide at the N-terminus, the peptide can remain on the support resin. The two cysteines are selectively deprotected prior to the addition of maleimide. Potentially catalyzed (heterogeneous) air oxidation can result in disulfides with peptides present on the resin. Maleimide is then added to the N-terminus and the peptide is cleaved from the resin and completely deprotected. A generalized reaction scheme for modification at an internal amino acid of a peptide containing two cysteines in a disulfide bridge is illustrated in the schematic diagram below.

[0225]

[Chemical 11] Alternatively, the peptide can be modified at internal amino acids (ie, neither the C-terminus nor the N-terminus). A generalized reaction scheme for modification at an internal amino acid of a peptide containing two cysteines in a disulfide bridge is illustrated in the scheme below.

[0226]

[Chemical 12] Examples of therapeutic peptides containing two cysteines as disulfide bridges include human osteocalcin 1-49, human diabetes related peptides, 5-28 fragment of human / dog atrial natriuretic peptide, bovine bactenecin (bact).
enecin), and human [Tyr0] -cortistatin (cortista)
tin) 29.

D. Peptides Containing Multiple Cysteines When the peptide contains multiple cysteines as disulfide bridges, the peptide is cleaved from the support resin prior to the addition of maleimide. For modification of the peptide from the C-terminus, all protecting groups carry a carboxy terminus (the carboxy terminus remains unprotected due to cleavage from the support resin) and two cysteines that are said to build a disulfide bridge. Excludes. Cysteines involved in other disulfide bridges are sequentially deprotected in pairs using selection of protecting groups. It is recommended to build and purify each crosslink one by one before moving on to the next crosslink. Mild air oxidation produces disulfide bridges, and peptides
It should be purified at each stage. Solution phase chemistry is then required to activate the C-terminus in the presence of the disulfide bridge and add the maleimide (via the amino-alkyl-maleimide) to the C-terminus. The peptide is then completely deprotected.

For modification of the peptide from the N-terminus, the peptide can be cleaved on a support resin, and selectively deprotecting the first two cysteines to build a disulfide under mild air oxidation. You can Subsequent deprotection is prior to addition of the maleimide,
Provides other disulfides. Air oxidation potentially promoted by catalysts (heterogeneous) can produce peptides and disulfides that are still on the resin. Maleimide is then added on the N-terminus and the peptide is cleaved from the resin and completely deprotected.

Alternatively, the peptide can be modified at internal amino acids (ie, neither at the C-terminus nor at the N-terminus).

Peptides containing multiple cysteines include human endothelin and [Lys
4] Sarafotoxin S6c is mentioned.

5. Administration of Modified Therapeutic Peptides The modified therapeutic peptides can be administered in a physiologically acceptable vehicle (eg, deionized water, phosphate buffered saline (PBS), saline). Water, aqueous ethanol or other alcohol, plasma, proteinaceous solution, mannitol, aqueous glucose, alcohol, vegetable oil, etc.). Other additives that may be included include buffers (the medium is generally buffered at a pH in the range of about 5-10, the buffers generally about 50
˜250 mM concentration range, salt (concentration of salt is generally about 5-500 mM)
, And physiologically acceptable stabilizers and the like. The composition may be lyophilized for convenient storage and shipping.

The modified therapeutic peptides are, for the most part,
It is administered orally, parenterally (eg, intravascularly (IV), intraarterially (IA), intramuscularly (IM), subcutaneously (SC), etc.). Administration will be by infusion, where appropriate. In some cases, where the reaction of the functional groups is relatively slow, administration may be oral, nasal, rectal, transdermal or aerosol, where the nature of the conjugate is , Allows transfer to the vascular system. Usually a single injection will be used, but more than one injection can be used if desired. The modified therapeutic peptide may be by any convenient means, including syringes, trocars, catheters, etc.
Can be administered by The particular mode of administration will vary depending on the amount to be administered, whether it is a single bolus, continuous administration, or the like. Preferably, administration is intravascular (the site of this introduction is not critical to the invention)
, Preferably where there is rapid blood flow (eg, intravenous, peripheral vein or central vein). Other routes may find use in which administration is associated with a delayed release technique or a protective matrix. This intent is for treatment (ltherapeuti
c) The peptide is efficiently distributed in the blood, so that it can react with blood components. Conjugate concentrations generally vary from about 1 pg / ml to 50 mg / ml and vary widely. The total amount administered intravascularly is generally about 0.1 mg / ml to about 10 mg / ml.
mg / ml, more usually in the range of about 1 mg / ml to about 5 mg / ml.

The binding of permanent components of blood such as immunoglobulins, serum albumin, red blood cells and platelets results in a number of advantages. The activity of the modified therapeutic peptide component is extended for days to weeks. Only one dose needs to be given during this period. Greater specificity may be achieved because the active compound is primarily bound to large molecules, where it is less likely to be taken up intracellularly so as to interfere with other physiological processes.

The formation of covalent bonds between blood components can occur in vivo or ex vivo. Modified therapeutics for ex vivo covalent bond formation
The peptide is added to blood, serum or saline solution containing human serum albumin or IgG to allow covalent bond formation between the modified therapeutic peptide and the blood component. In a preferred manner, the therapeutic peptide is modified with maleimide and reacted with human serum albumin in saline solution. Once the modified therapeutic peptide reacts with blood components to form a therapeutic peptide protein conjugate, the conjugate can be administered to a patient.

Alternatively, the modified therapeutic peptide can be administered directly to the patient, such that
A covalent bond forms in vivo between the modified therapeutic peptide and the binding moiety.

Furthermore, if localized delivery of a therapeutic peptide is desired, several delivery methods can be used.

A. Lavage of Incision Surgery Area There are numerous applications for topical therapeutic compounds, including administration of therapeutic compounds as an adjunct to open surgery. In these cases, the therapeutic compound is irrigated at the surgical site (ie, a portion of that site) prior to closure, or the therapeutic compound is treated in a confined space (eg, after an endarterectomy procedure). Incubated in the arterial section or part of the GI tract during ablation) for a short period of time, and excess fluid is subsequently removed.

B. Incubation of Tissue Graft Tissue grafts (eg, autologous and xenobiotic vein / arterial grafts and valve grafts and organ grafts) have been modified with therapeutic compounds. It may be possible to allow the covalent bond to form by either pretreating it and incubating the implant in a therapeutic solution and / or perfusing the implant with such a solution.

C. Catheter Delivery Catheters may be used as part of an endoscopic procedure inside an organ (eg, bladder, GI tract, vagina / uterus) or as a cardiovascular catheter procedure (eg, balloon angioplasty). It is used to deliver therapeutic compounds, either as an adjunct to surgery). Standard catheters and newer drug delivery and iontophoresis catheters can be utilized.

D. Direct Injection For certain poorly vascularized spaces (eg, intra-articular joint space), direct injection of a therapeutic compound bioconjugates to superficial tissue.
And obtain the desired duration of drug effect. Other indications may include: intramedullary injection, intratumoral injection, intravaginal injection, intrauterine injection, enteral injection, Eustachioinjection, intrathecal injection, subcutaneous injection, intraarticular injection, intraperitoneal injection or ocular. Via internal injection as well as via the bronchoscope, via the nasogastric tube and via nephrostomy.

6. Monitoring the Presence of Modified Therapeutic Peptide Derivatives Another aspect of the invention is the therapeutic peptides and / or analogs in biological samples (eg, blood), or their derivatives and binding. It relates to a method for determining body concentration, as well as for determining the peptidase stability of modified peptides. The blood of a mammalian host can be monitored one or more times for the presence of the modified therapeutic peptide compound. By taking a portion or sample of the host's blood, it may be determined whether the therapeutic peptide is bound to a permanent blood component in an amount sufficient to be therapeutically active, and then treatment in the blood. The level of the peptide component of interest can be determined. If desired, it can also be determined to which blood component the therapeutic peptide derivative molecule is bound. This is especially important when using non-specific therapeutic peptides. Calculating serum albumin and IgG half-lives for a particular maleimide therapeutic peptide is much easier.

One method of determining the concentration of therapeutic peptides, analogs, derivatives and conjugates is to use antibodies specific for the therapeutic peptides or therapeutic peptide analogs or their derivatives and conjugates, and Therapeutic peptides such as
Use of the antibody as a treatment for toxicity potentially associated with analogs, and / or their derivatives or conjugates. This is due to the increased stability and longevity of therapeutic peptides in vivo in patients, which is a new problem during treatment (
(Including increased potential for toxicity). However, in some cases it should be mentioned that conventional antibody assays may not recognize the difference between the cleaved and uncleaved therapeutic peptides. In such cases, other assay techniques may be used (eg LC / MS) (liquid chromatography / mass spectrometry).

The use of antibodies (either monoclonal or polyclonal) with specificity for a particular therapeutic peptide, analog or derivative thereof may aid in mediating any such issues. Antibodies are generated from a host immunized with a particular therapeutic peptide, analog or derivative thereof, or with an immunogenic fragment of the factor, or a synthetic immunogen corresponding to an antigenic determinant of the factor. Can be or be derived from. Preferred antibodies have high specificity and affinity for the native, derivatized, and conjugated forms of therapeutic peptides or therapeutic peptide analogs. Such antibodies can also be labeled with enzymes, fluorescent dyes, or radioactive labels.

Antibodies specific for derivatized therapeutic peptides can be produced by using the purified therapeutic peptides for the induction of derivatized therapeutic peptide-specific antibodies. The induction of antibodies contemplates not only stimulation of the immune response by injection into animals, but also similar steps in the production of synthetic antibodies or other specific binding molecules (eg, screening of recombinant immunoglobulin libraries). . Both monoclonal and polyclonal antibodies can be produced by procedures well known in the art. In some cases, the use of monoclonal antibodies may be preferable to polyclonal antibodies (eg, where degradation occurs over a region beyond epitope / antibody recognition).

Antibodies can be used to treat toxicity induced by the administration of therapeutic peptides, analogs or derivatives thereof, and can be used ex vivo or in vivo. The ex vivo method involves immunodialysis treatment for toxicity using antibodies immobilized on a solid support. In vivo methods include the administration of an amount of antibody effective to induce clearance of the antibody-drug complex.

Antibodies can be used to remove therapeutic peptides, analogs or derivatives thereof, and conjugates thereof from blood of patients ex vivo by contacting the blood with the antibody under sterile conditions. For example, the antibody may be immobilized on the column matrix (
fix) or otherwise immobilized,
The patient's blood can then be drawn from the patient and passed through the matrix. Blood in which the therapeutic peptide analog, derivative or conjugate binds to the antibody and low concentrations of the therapeutic peptide, analog, derivative or conjugate can then be returned to the patient's circulatory system. The amount of therapeutic peptide compound removed can be controlled by adjusting the pressure and flow rate. Preferential removal of therapeutic peptides, analogs, derivatives and conjugates from plasma components of the patient's blood has been described, for example:
It can be provided by the use of a semipermeable membrane or otherwise by first separating the plasma component from the cellular component by methods known in the art before the plasma component passes through the matrix containing the anti-therapeutic antibody. . Alternatively, preferential removal of therapeutic peptide-conjugated blood cells (including red blood cells) collects and concentrates blood cells in the patient's blood as well as their elimination relative to the elimination of serum components of the patient's blood. It can be provided by contacting the cells with an immobilized anti-therapeutic antibody.

The antibody may be administered parenterally in vivo to a patient receiving a therapeutic peptide, analog, derivative or conjugate for treatment. The antibody binds to the therapeutic peptide compound and the conjugate. Once bound to the therapeutic peptide, activity is hindered, if not completely blocked, thereby reducing the biologically effective concentration of the therapeutic peptide compound in the patient's bloodstream, and adverse side effects. To minimize. In addition, the bound antibody-therapeutic peptide complex facilitates clearance of the therapeutic peptide compound and conjugate from the patient's bloodstream.

The fully described invention is now illustrated by the following non-limiting examples.

EXAMPLES A. General Method for Synthesis of Modified Therapeutic Peptides Solid phase peptide synthesis of the modified peptides on a 100 μmol scale was performed using Fm
oc protected Rink Amide MBHA resin, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU) N, N-dimethylformamide (DM).
F) Sympho with activation in solution and N-methylmorpholine (NMM)
Performed on ny Peptide Synthesizer and performed piperidine deprotection of the Fmoc group (step 1). Deprotection of the terminal Fmoc group was achieved with 20% piperidine (step 2), followed by 3-maleimidopropionic acid (
3-MPA) coupling, acetic acid coupling or one or more Fm
Either oc-AEEA coupling is performed, followed by 3-MPA coupling (step 3). Cleavage of the resin and isolation of the product was performed with 86% TFA / 5.
% TIS / 5% with H ₂ O / 2% thioanisole and 2% phenol executed, followed by precipitation by dry-ice cold Et ₂ O (Step 4).
The product was applied to Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 m
m × 25 cm column and UV detector (Varian Dynamax UVD
II) Dynamax C ₁₈ , 60Å with (λ214 and 254 nm)
Purify by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system using an 8 μm, 21 mm × 25 cm column.
The product is a Hewlett Packard equipped with a diode array detector.
It should have a purity of> 95% as determined by RP-HPLC mass spectrometry using a LCMS-1100 series spectrometer and using electrospray ionization.

B. Alteration of Native Peptide Chain To facilitate peptide modification, one or more amino acid residues were added to Examples 1-5.
Can be added to the peptide and / or one or more amino acid residues can be replaced with other amino acid residues. This modification aids in the attachment of reactive groups.

Example 1 Addition of Lys at the C-terminus of Kringle-5. NAc-Pro-A
rg-Lys-Leu-Tyr- Asp-Tyr-Lys-NH 2. Preparation of 3TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide:
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH. Deblocking the Fmoc group at the N-terminus of resin-bound amino acids was carried out using 20% in DMF.
% Piperidine for about 15-20 minutes. Acetic acid coupling was performed under conditions similar to amino acid coupling. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Example 2 Addition of Lys at the C-terminus of Kringle-5. NAc-Arg-L
ys-Leu-Tyr-Asp- Tyr-Lys-NH 2. 2TFA. 3TFA
The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH. The reverse block of the Fmoc group at the N-terminus of the resin bound amino acid was reduced to about 1 with 20% piperidine in DMF.
Run for 5-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Example 3 Addition of Lys at the N-terminus of Kringle-5. NAc-Tyr-T
hr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-T
Preparation of yr-Lys-NH2.3TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fm
oc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc
-Thr (tBu) -OH, Fmoc-Tyr (tBu) OH. Deblocking of the Fmoc group at the N-terminus of the resin-bound amino acid was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Example 4 Addition of Lys at the N-terminus of Kringle-5. Position 5 of Cys
Substitution with Ala at 24. NAc-Arg-Asn-Pro-Asp-Gl
y-Asp-Val-Gly-Gly-Pro-Trp-Ala ⁵²⁴ -Tyr-
Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-
Tyr-Lys-NH _2. Preparation of 4TFA) The following protected amino acids were linked to Rink Amide using an automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fm
oc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc
-Thr (tBu) -OH, Fmoc-Tyr (tBu) OH, Fmoc-Al
a-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gl
y-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-As
p (OtBu) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Pr
o-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf)-
OH. The reverse block of the Fmoc group at the N-terminus of the resin-bound amino acid was replaced with DMF.
Run for about 15-20 minutes with 20% piperidine in. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Example 5 Addition of Lys at the N-terminus of Kringle-5. NAc-Ar
g-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly-Pr
_{o-Trp-Lys-NH 2} . Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fm
oc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly
-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (
Pbf) -OH. Deblocking of the Fmoc group at the N-terminus of the resin bound amino acid was performed with 20% piperidine in DMF for 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

[0256]   (Example 6 Preparation of D-Ala2 GLP-1 (7-36) amide)   Solid phase peptide synthesis of GLP-1 analogs on a 100 μmol scale was performed manually.
Solid phase synthesis and Fmoc protected Rink Amide MBHA resin, Fmo
c-protected amino acid, O-benzotriazol-1-yl-N, N, N ', N'-te
Tramethyl-uronium hexafluorophosphate (HBTU) N, N-di
Methylformamide (DMF) solution and N-methylmorpholine (NMM)
Symphony Peptide Synthesizer with activation
Was carried out and the piperidine deprotection of the Fmoc group was performed (step 1). resin
Cleavage and isolation of the product was performed using 85% TFA / 5% TIS / 5% thioaniline.
Performed with sole and 5% phenol, followed by dry ice cooled Et ₂ Precipitate with O (step 2). Varian (Rainin) preparation of product
Binary HPLC system (: 30-55% B (H₂0.045% in O
TFA (A) and CH₃Gradient elution of 0.045% TFA (B)) in CN)
With Phenomenex Luna 10μ phenyl-hexyl, 2
1 mm x 25 cm column and UV detector (Varian Dynamax U
Preparative reverse phase HPLC using VD II) (λ214 and 254 nm).
The desired peptide was purified by RP-HP for 180 minutes at 9.5 mL / min.
Obtained with> 95% purity as determined by LC. These steps are outlined below.
It is shown schematically in the diagram.

[0257]

[Chemical 13] C. Preparation of Modified Peptides from Cysteine-Free Peptides The preparation of maleimide peptides from therapeutic peptides containing multiple protected functional groups and containing no cysteine was performed by synthesizing the peptides as described below. Is illustrated by. The peptide may be modified at the N-terminus, the C-terminus or at the amino acid located between the N- and C-termini. The modified peptide is synthesized by cleaving off the N-terminus of the native peptide sequence or the modified C-terminus of the native peptide sequence. One or more additional amino acids may be added to the therapeutic peptide to facilitate attachment of the reactive group.

(1. Modification of therapeutic peptide at the C-terminus) (Example 7) Modification of RSV peptide at ε-amino group of added C-terminal lysine residue Val-Ile-Thr-Ile-Glu-Leu- Ser-Asn
-Ile-Lys-Glu-Asn-Lys-Met-Asn-Gly-Ala
-Lys-Val-Lys-Leu-le-Lys-Gln-Glu-Leu
-Asp-Lys-Tyr-Lys-Asn-Ala-Val-Lys- (Nε
-Preparation of MPA). ) Solid phase peptide synthesis of DAC analogs on a 100 μmol scale was performed using manual solid phase synthesis, Symphony Peptide Synthesizer and Fm.
oc protection Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Lys (Aloc) -OH, Fmo.
c-Val-OH, Fmoc-Ala-OH, Fmoc-Asn (Trt) -O
H, Fmoc-Lys (Boc) -OH, Fmoc-Tyr (tBu) -OH,
Fmoc-Lys (Boc) -OH, Fmoc-Asp (tBu) -OH, Fm
oc-Leu-OH, Fmoc-Glu (tBu) -OH, Fmoc-Gln (
Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-le-OH
, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-V
al-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, F
moc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Met
-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asn (Trt) -O
H, Fmoc-Glu (tBu) -OH, Fmoc-Lys (Boc) -OH,
Fmoc-le-OH, Fmoc-Asn (Trt) -OH, Fmoc-Se
r (tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (tBu)-
OH, Fmoc-Ile-OH, Fmoc-Thr (tBu) -OH, Fmoc
-Ile-OH, Fmoc-Val-OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazole-
Activation with 1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by 20% (V / V) piperidine N, N-
Activate with a dimethylformamide (DMF) solution for 20 minutes (step 1).
Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHC
3 equivalents of Pd (PP dissolved in 13: NMM: HOAc (18: 1: 0.5)
This is achieved by treating the resin with the solution of h3) 4 for 2 hours (step 2). The resin was then loaded with CHCl3 (6 x 5 mL), 20% HOAc in DCM (6
X5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide was cleaved from the resin with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cooled Et2O.
(Step 4). The product, Varian (Rainin) binary HPLC system for preparing _{(: 30~55% B (H 2} O 0.045% in T
Using FA Gradient elution (A) and CH ₃ CN 0.045% in TFA (B))), the Phenomenex Luna 10 [mu] phenyl - hexyl, 21
mm × 25 cm column and UV detector (Varian Dynamax UV
Purify by preparative reverse-phase HPLC at 9.5 mL / min for 180 min with D II) (λ214 and 254 nm) to give the desired DAC in> 95% purity as determined by RP-HPLC. .

Example 8 Dyn A in the ε-amino group of an added C-terminal lysine residue
Modification of 1-13-Dyn A 1-13 (Nε-MPA) -NH ₂ Tyr-G
ly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pro-L
Synthesis of ys-Leu-Lys- (Nε-MPA) -NH ₂ A solid phase peptide synthesis of Dyn A 1-13 on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. Use and execute. The following protected amino acids are sequentially added to the resin: Fmoc-Lys (Aloc) -OH,
Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pr
o-OH, Fmoc-Arg (Pbf) -OH, Fmoc-le-H, Fmo
c-Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-
Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-
Gly-OH, Fmoc-Tyr (tBu) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uronium hexafluorophosphate (HBTU). ) And diisopropylethylamine (DIEA)
To activate. Removal of the Fmoc protecting group is accomplished with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) in 20 minutes (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL
3 equivalents of P dissolved in CHCl ₃ : NMM: HOAc (18: 1: 0.5)
Achieved by treating the resin with a solution of d (PPh ₃ ) ₄ for 2 hours (step 2)
. The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOA in DCM.
Washed with c (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide was added to 85% TFA / 5% TIS / 5% thioanisole and 5
% Of phenol and cleaved from resin followed by dry ice cooled E
Precipitate with t ₂ O (step 4). The product is Varian (Rainin)
Preparative Binary HPLC System (: 30-55% B (0.045 in H ₂ O
% TFA (A) and gradient elution of 0.045% TFA (B) in CH ₃ CN) was used with Phenomenex Luna 10 μ phenyl-hexyl, 21 mm × 25 cm column and UV detector (Varian Dynamax).
Preparative reverse phase HPLC using UVD II) (λ214 and 254 nm).
Purify for 180 min at 9.5 mL / min to give the desired DAC in> 95% purity as determined by RP-HPLC.

[0260]   The structure of this product is:

[0261]

[Chemical 14] Is.

Example 9 Dyn A at the ε-amino group of an added C-terminal lysine residue
Modification of 2-13-Dyn A 2-13 (Nε-MPA) -NH ₂ Gly-G
ly-Phe-Leu-Arg-Arg-lle-Arg-Pro-Lys-L
Synthesis of eu-Lys- (Nε-MPA) -NH ₂ ) Using automated peptide synthesis, the following protected amino acids were sequentially added to Link Am.
added to ide MBHA resin: Fmoc-Lys (Mtt) -OH, Fmo
c-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-O
H, Fmoc-Arg (Pbf) -OH, Fmoc-lle-OH, Fmoc-
Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Le
u-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Boc-
Gly-OH. Manual synthesis was used for the remaining steps: HBTU in DMF.
Selective removal of Mtt group and coupling of MPA with / HOBt / DIEA activation. The target dynorphin analog was removed from the resin; the product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid on lyophilization in 35% yield. Analytical HPLC showed that the product had R _t = 3.
It was shown to be> 95% pure at 0.42 minutes. ESI-MS m / z: C ₇₃ H _{123
^{N 25 O 15 (MH +)}} , calcd 1590.0, found MH ³⁺ 531.3.

Example 10 Dyn at the ε-amino group of the added C-terminal lysine residue
Modification of A 1-13-Dyn A 1-13 (AEA ₃ -MPA) -NH ₂ Ty
r-Gly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pr
o-Lys-Leu-Lys- ( AEA 3 -MPA) using synthetic) automated peptide synthesis -NH _2, the following protected amino acids, continuously Rink Am
added to ide MBHA resin: Fmoc-Lys (Mtt) -OH, Fmo
c-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-O
H, Fmoc-Arg (Pbf) -OH, Fmoc-lle-OH, Fmoc-
Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Le
u-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gl
y-OH and Boc-Tyr (Boc) -OH. Manual synthesis was used for the remaining steps: selective removal of the Mtt group, 3 Fmo between each coupling.
Coupling with c-AEA-OH group (AEA = aminoethoxyacetic acid) with Fmoc removal and MP with HBTU / HOBt / DIEA activity in DMF.
A acid. The target dynorphin analog was removed from the resin; the product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid on lyophilization in 29% yield. Analytical HPLC showed that the product had R _t = 33.
It was shown to be> 95% pure at 06 minutes. ESI-MS m / z: C ₉₄ H ₁₅₄ N ₂₉
O ₂₃ (MH ⁺ ), calculated value 2057.2, measured value MH ⁴⁺ 515.4, MH ³⁺
686.9, MH ²⁺ 1029.7.

[0264]

[Chemical 15]   Example 11 Dyn at ε-amino group of added C-terminal lysine residue
Modification of A 2-13-Dyn A 2-13 (AEA₃-MPA) -NH₂  GI
y-Gly-Phe-Leu-Arg-Arg-le-Arg-Pro-Ly
s-Leu-Lys- (AEA₃-MPA) -NH₂Of)   The following protected amino acids were sequentially added to the Link Am using automated peptide synthesis.
added to ide MBHA resin: Fmoc-Lys (Mtt) -OH, Fmo
c-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Pro-O
H, Fmoc-Arg (Pbf) -OH, Fmoc-lle-OH, Fmoc-
Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Le
u-OH, Fmoc-Phe-OH, Fmoc-Gly-OH and Fmoc-
Gly-OH. Manual synthesis was used for the remaining steps: Selective removal of the Mtt group.
And 3 Fmoc-AEA-OH groups and Fmoc removal between each coupling.
Coupling with HBTU / HOBt / DIEA activity in DMF
MPA used. The target dynorphin analog was removed from the resin; the product
Isolated by precipitation and purified by preparative HPLC to give 29
The desired product was obtained as a white solid in% yield. Analytical HPLC showed that the product was R_t
= 31.88 mins indicated> 95% purity. ESI-MS m / z: C₈₅H ₁₄₅ N_{twenty five}O_{twenty one}(MH⁺), Calculated value 1894.3, measured value MH⁴⁺  474.6, M
H³⁺  632.4, MH²⁺  948.10.

[0265]

[Chemical 16] Example 12 Modification of Neuropeptide Y at ε-Amino Group of Added C-Terminal Lysine Residue-Tyr-Pro-Ser-Lys-Pro-Asp-As
n-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-As
p-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Ly
Preparation of s- (N-εMPA) -NH ₂ ) Solid phase peptide synthesis of a modified neuropeptide Y analog on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synt.
Run with hesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Lys (
Aloc) -OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmo
c-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-
Tyr (tBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Al
a-OH, Fmoc-Met-OH, Fmoc-Asp (tBu) -OH, Fm
oc-Glu (tBu) -OH, Fmoc-Ala-OH, Fmoc-Pro-
OH, Fmoc-Ala-OH, Fmoc-Asp (tBu) -OH, Fmoc
-Glu (tBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH
, Fmoc-Asn (Trt) -OH, Fmoc-Asp (tBu) -OH, F
moc-Pro-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ser
(TBu) -OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -O
H. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uronium hexafluorophosphate (HBTU). ) And diisopropylethylamine (DIEA). Removal of the Fmoc protecting group
Activation is performed using a 20% (V / V) piperidine solution in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0).
． Achieved by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in 5) for 2 hours (step 2). The resin was then added to CHCl ₃ (6 × 5 mL
), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide was added to 85% TFA / 5% TIS /
Cleavage from the resin with 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product
Varian (Rainin) Preparative Binary HPLC System (: 30-5)
5% B (0.045% TFA (A) in H ₂ O and 0.045% in CH ₃ CN
(% TFA (B)) gradient elution) with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (V
Arian Dynamax UVD II) (λ214 and 254 nm) was used to purify by preparative reverse phase HPLC at 9.5 mL / min for 180 min and the desired DAC in> 95% purity as determined by RP-HPLC. To get

Example 13 Modification of GLP-1 (7-36) -C in C-Terminal Arginine-G
Preparation of LP-1 (7-36) -EDA-MPA) Solid phase peptide synthesis of modified GLP-1 analogs on a 100 μmol scale was performed manually and with Symphony Peptide Synthesizer.
Run on SASRIN (super acid sensitive resin). The following protected amino acids are sequentially added to the resin: Fmoc-Arg (Pbf) -OH, Fmoc-Gly-O.
H, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-
Leu-OH, Fmoc-Trp (Boc) -OH, Fmoc-Ala-OH,
Fmoc-le-OH, Fmoc-Phe-OH, Fmoc-Glu (OtB
u) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, F
moc-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Gly
-OH, Fmco-Glu (OtBu) -OH, Fmoc-Leu-OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc
-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (tB
u) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu)
-OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmo
c-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-
OH, Boc-His (Trt) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazole-1
Activation with -yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). The fully protected peptide is cleaved from the resin by treatment with 1% TFA / DCM (step 2). Then ethylenediamine and 3-maleimidopropionic acid are sequentially added to the free C-terminus (step 3). The protecting groups were then cleaved and the product isolated with 86% TFA / 5% TIS / 5% H ₂ O / 2% thioanisole and 2% phenol, followed by dry ice. Precipitate with chilled Et ₂ O (step 4). The product was
C _18, Dynamax C ₁₈ with 60 Å, 8 [mu] m guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax UVD II) (λ214 and 254nm), 60Å, 8μm, 21mm × 25
Purify by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system with a cm column to obtain the desired DAC in> 95% purity as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

[0267]

[Chemical 17] Example 14 Modification of exendin-4 at the C-terminal serine. Preparation of exendin-4 (1-39) -EDA-MPA Solid phase peptide synthesis of modified exendin-4 analog at 100 μmol scale. , Manually and Symphony Peptide Synthesi
run on zer SASRIN (super acid sensitive resin). The following protected amino acids are sequentially added to the resin: Fmoc-Ser (tBu) -OH, Fmoc-P.
ro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-A
la-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, F
moc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gly
-OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmo
c-Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Trp (B
oc) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-le-OH
, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Arg (Pb
f) -OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-G
lu (OtBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-G
lu (OtBu) -OH, Fmoc-Met-OH, Fmoc-Gln (Trt
) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Ser (tBu)-
OH, Fmoc-Leu-OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-
Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH,
Fmoc-Glu (OtBu) -OH, Fmoc-Gly-OH, Boc-Hi
s (Trt) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N
', N'-Tetramethyl-uronium hexafluorophosphate (HBTU)
And activated with diisopropylethylamine (DIEA). Fmoc
Removal of the protecting groups is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). The fully protected peptide is cleaved from the resin by treatment with 1% TFA / DCM (step 2
). Then ethylenediamine and 3-maleimidopropionic acid are added to the free C
Add to the ends continuously (step 3). The protecting groups were then cleaved and the product isolated with 86% TFA / 5% TIS / 5% H ₂ O / 2% thioanisole and 2% phenol, followed by dry ice. Chilled Et ₂
Precipitate with O (step 4). The product was treated with Dynamax C ₁₈ , 60Å, 8
μm guard module, 21 mm × 25 cm column and UV detector (Var
ia Dynamax UVD II) (λ214 and 254 nm) with a Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column using a Varian (Rainin) preparative binary HPLC system, purified by preparative reverse phase HPLC and RP. > 9 as determined by HPLC
The desired DAC is obtained with 5% purity.

[0268]

[Chemical 18] Example 15 Modification of the secretin peptide at the epsilon amino group of the added C-terminal lysine residue. His-Ser-Asp-Gly-Thr-Phe-Thr-
Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-
Ala-Arg-Leu-Glu-Arg-Leu-Leu-Gln-Gly-
Leu-Val-Lys- (Nε- MPA) Preparation of -NH ₂₎ 100 [mu] mol of a scale, modified solid phase peptide synthesis of secretin peptide analog manual solid-phase synthesis, Symphony Peptide Synth
Run with ezizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Lys (A
loc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc
-Gly-OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH
, Fmoc-Leu-OH, Fmoc-Arg (Pbf) -OH, Fmoc-G
lu (tBu) -OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf)
-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Glu
(TBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Leu-O
H, Fmoc-Arg (Pbf) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Leu-OH, Fmoc-Glu (tBu) -OH, Fmoc-Se
r (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-
OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc
-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-H
is (Boc) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N,
N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU
) And diisopropylethylamine (DIEA). Fmo
Removal of the c-protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Lys (Aloc)
Selective deprotection of groups was performed manually and 5 mL of CHCl ₃ : NMM: HO
This is accomplished by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in Ac (18: 1: 0.5) for 2 hours (step 2). Then, this resin is changed to CH
Cl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL), DCM (
6 × 5 mL) and DMF (6 × 5 mL). This synthesis is then
Re-automate for the addition of 3-maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. 85% TF
Cleavage from the resin with A / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product, Varian (Rainin) binary HPLC system for preparing _{(: 30~55% B (H 2} 0.045% TFA (A in O) and CH ₃ CN 0.045% in TFA in (B)) Gradient elution)
18 by preparative reverse phase HPLC at 9.5 mL / min using a Nex Luna 10 [mu] Phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) ([lambda] 214 and 254 nm).
Purify over 0 minutes to obtain the desired DAC in> 95% purity as determined by RP-HPLC.

Example 16 Modification of kringle-5 at the ε-amino group of the added C-terminal lysine residue. NAc-Pro-Arg-Lys-Leu-Tyr-Asp-T.
yr-Lys- (Nε-MPA) -NH 2. Preparation of 2TFA) Solid phase peptide synthesis of modified kringle-5 peptide at 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synthes.
It was carried out with an izer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Lys (Alo
c) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Asp (tBu)
-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmo
c-Lys (Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-
Pro-OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N
Activation with'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by removing 20% (V / V) piperidine in N, N-dimethylformamide (DM).
F) Activate with solution for 20 minutes (step 1). At the end of the synthesis acetic anhydride,
Added to acetylate the N-terminus. Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl ₃ : NMM: HOAc (18: 1:
This is accomplished by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in 0.5) for 2 hours (step 2). This resin was then treated with CHCl ₃ (6 × 5 m
L), washed with 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide was added to 85% TFA / 5% TIS
Cleave from the resin with / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-
_{55% B (H 2 O 0.045} % in TFA (A) and CH ₃ 0.04 in CN
5% TFA (B)) gradient elution) was used with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Purify by reverse phase preparative HPLC at 9.5 mL / min for 180 min to give the desired DAC in> 95% purity as determined by RP-HPLC.

Example 17 Modification of Kringle-5 at the ε-amino group of the added C-terminal lysine residue. NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Ly.
s-Leu-Tyr-Asp- Tyr-Lys- (Nε-MPA) -NH 2. Two
Preparation of TFA) The following protected amino acids were sequentially added to the Rink Amide MBHA resin using automated peptide synthesis: Fmoc-Lys (Aloc) -OH, F.
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, F
moc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmo
c-Thr (tBu) -OH, Fmoc-Tyr (tBu) OH (step 1). The reverse block of the Fmoc group at the N-terminus of the resin bound amino acid was reduced to 20% in DMF.
Was carried out for about 15 to 20 minutes. The final cleavage from the resin was performed with the cleavage mixture as described above. The product is isolated by precipitation and H
Purify by PLC to give the desired product as a white solid upon lyophilization. Ly
Selective deprotection of the s (Aloc) group was carried out manually and 5 mL of CHCl ₃
: NMM: HOAc (18: 1: 0.5) dissolved in 3 equivalents of Pd (PPh ₃
) By treating the resin with the solution of ₄ for 2 hours (step 2). Then
This resin was treated with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). Resin cleaving and product isolation 85% TFA / 5% TIS / 5%
Carried out with thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product is Varian
(Rainin) Preparative Binary HPLC System (: 30-55% B (H ₂
0.045% TFA (A) in O and 0.045% TFA in CH ₃ CN
(B)) gradient elution) was used, Phenomenex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian).
Purified by preparative reverse phase HPLC using Dynamax UVD II) (λ214 and 254 nm) at 9.5 mL / min for 180 min.

Example 18 Modification of the kringle at the ε-amino group of the added C-terminal lysine residue. NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-
Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-
Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys- (Nε-
MPA) -NH _2. Preparation of 3TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, F
moc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmo
c-Thr (tBu) -OH, Fmoc-Tyr (tBu) OH, Fmoc-A
la-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-G
ly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-A
sp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtB
u) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, F
moc-Arg (Pbf) -OH (step 1). N-terminal Fm of resin-bound amino acid
Deblocking of the oc group was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the coupling of amino acids. The final cleavage from the resin was performed with the cleavage mixture as described above.
The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of 3-maleimido prepionic acid (step 3). Resin cleavage and product isolation was performed using 85% TFA / 5% TIS /
Performed with 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product is
an (Rainin) preparative binary HPLC system (: 30-55% B (
0.045% TFA (A) in H ₂ O and 0.045% TF in CH ₃ CN
A (B)) gradient elution) was used with Phenomenex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varia).
n Dynamax UVD II) (λ214 and 254 nm)
Purified by preparative reverse phase HPLC at 9.5 mL / min for 180 minutes.

Example 19 Modification of Kringle-5 at the ε-amino group of the added C-terminal lysine residue. NAc-Arg-Asn-Pro-Asp-Gly-Asp-Va.
l-Gly-Gly-Pro- Trp-Lys- (Nε-MPA) -NH 2.
Preparation of TFA) The following protected amino acids were sequentially added to the Rink Amide MBHA resin using automated peptide synthesis: Fmoc-Lys (Aloc) -OH, F.
moc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, F
moc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu
) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, F
moc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg
(Pbf) -OH (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0.5). This was achieved by treating the resin for 2 hours at (step 2). The resin was then treated with CHCl ₃ (6 × 5 mL), DC
20% HOAc in M (6 x 5 mL), DCM (6 x 5 mL), and DMF.
Wash with (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimido prepionic acid (step 3). Resin cleavage and product isolation was performed using 85
Performed with% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product, Varian (Rainin) binary HPLC system for preparing _{(: 30~55% B (H 2} 0.045% TFA (A in O) and CH ₃ CN 0.045% in TFA in (B)) Gradient elution)
18 by preparative reverse phase HPLC at 9.5 mL / min using a Nex Luna 10 [mu] Phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) ([lambda] 214 and 254 nm).
Purified over 0 minutes.

Example 20 Modification of kringle-5 at the epsilon amino group of the added C-terminal lysine residue. NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Ly.
_{s- (Nε-MPA) -NH 2} . 2TFA Preparation) Using automated peptide synthesis, the following protected amino acids were linked to the Link Amide:
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH (step 1). Deblocking of the Fmoc group at the N-terminus of the resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the coupling of amino acids. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization. Lys (Al
oc) group selective deprotection was performed manually, and 5 mL of CHCl ₃ : NMM
: HOAc (18: 1: 0.5) was achieved by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL), D
Wash with CM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimido prepionic acid (step 3). Resin cleavage and product isolation was carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Rain
in) binary HPLC system for preparing (: 30~55% B (0 in H ₂ O.
With 045% TFA (A) and CH ₃ CN 0.045% in TFA (B) gradient elution)), phenyl Phenomenex Luna 10 [mu] - hexyl, 21 mm × 25 cm column and UV detector (Varian Dyna
max UVD II) (λ 214 and 254 nm) using preparative reverse phase H
Purified by PLC at 9.5 mL / min for 180 minutes.

Example 21 Modification of Kringle-5 at the ε-amino group of the added C-terminal lysine residue. NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Ly.
_{s- (Nε-MPA) -NH 2} . 2TFA Preparation) Using automated peptide synthesis, the following protected amino acids were linked to the Link Amide:
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) -OH, Fm
oc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (
Pbf) -OH, Fmoc-Pro-OH (step 1). Reverse blocking at the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the coupling of amino acids. The final cleavage from the resin was performed with the cleavage mixture as described above. The product was isolated by precipitation and purified by HPLC to give the desired product as a white solid on lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of 3-maleimido prepionic acid (step 3). Resin cleavage and product isolation was performed using 85% TFA / 5% TIS /
Performed with 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product is
an (Rainin) preparative binary HPLC system (: 30-55% B (
0.045% TFA (A) in H ₂ O and 0.045% TF in CH ₃ CN
A (B)) gradient elution) was used with Phenomenex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varia).
n Dynamax UVD II) (λ214 and 254 nm)
Purified by preparative reverse phase HPLC at 9.5 mL / min for 180 minutes.

Example 22 Modification of Kringle-5 at the ε-amino acid group of the added C-terminal lysine residue. NAc-Pro-Arg-Lys-Leu-Tyr-Asp-
Tyr-Lys- (Nε-AEEA- MPA) -NH 2. 2TFA Preparation) Using automated peptide synthesis, the following protected amino acids were linked to the Link Amide:
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (
Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH
(Step 1). The reverse block of the Fmoc group at the N-terminus of the resin-bound amino acid was replaced by D
Performed for about 15-20 minutes with 20% piperidine in MF. The coupling of acetic acid was performed under similar conditions to the coupling of amino acids. Lys (Alo
c) Selective deprotection of the group was performed manually and 5 mL of CHCl ₃ : NMM:
This was accomplished by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ in HOAc (18: 1: 0.5) for 2 hours (step 2). Then, this resin
CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL), DC
Wash with M (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for addition (step 3). The synthesis was then re-automated for the addition of AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimidopropionic acid (MPA) (step 3). 85% for resin cleavage and product isolation
Performed with TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-55% B (0.045% TFA (A) and CH ₃ C in H ₂ O).
Gradient elution of 0.045% TFA (B)) in N).
180 by preparative reverse phase HPLC at 9.5 mL / min using ex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) (λ214 and 254 nm).
Purified over minutes.

Example 23 Modification of Kringle-5 at the ε-amino group of the added C-terminal lysine residue. NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Ty.
_{r-Lys- (Nε-AEEA n} -MPA) -NH 2. 2TFA Preparation) Using automated peptide synthesis, the following protected amino acids were linked to the Link Amide:
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (
Boc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH
(Step 1). The reverse block of the Fmoc group at the N-terminus of the resin-bound amino acid was replaced by D
Performed for about 15-20 minutes with 20% piperidine in MF. The coupling of acetic acid was performed under similar conditions to the coupling of amino acids.

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
This synthesis was re-automated for addition. The synthesis was then re-automated for the addition of 3-maleimido prepionic acid (MPA) and AEEA (aminoethoxyethoxy acetic acid) groups (step 3). 85% for resin cleavage and product isolation
Performed with TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4
). The product, Varian (Rainin) binary HPLC system for preparing (: 0.045% TFA (A of 30~55% B (H ₂ in O) and CH ₃ CN
Gradient elution of 0.045% TFA (B)) in
x Luna 10 [mu] Phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) ([lambda] 214 and 254 nm) were used to purify for 180 min at 9.5 mL / min by preparative reverse phase HPLC.

Example 24 GLP-1 at the ε-amino group of the added C-terminal lysine residue
Modification of. ^{GLP-1 (1-36) -Lys 37} (Nε-MPA) -NH 2. 5TF
A; His-Asp-Glu-Phe-Glu-Arg-His-Ala-Gl
u-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Se
r-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Gl
u-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Ar
g-Lys (Nε-MPA) -NH 2. Preparation of 5TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fm
oc-Trp-OH, Fmoc-Ala-OH, Fmoc-le-OH, Fm
oc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fm
oc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (
OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Pbf) -O.
H, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmo
c-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (N
-Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (O
tBu) -OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -O
H, Fmoc-Asp (OBu) -OH, Boc-His (N-Trt) -OH
(Step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated due to the addition of 3-maleimido prepionic acid (MPA) (step 3). Resin cleavage and product isolation using 85% TFA / 5%
Performed with TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-
_{55% B (H 2 O 0.045} % in TFA (A) and CH ₃ 0.04 in CN
5% TFA (B)) gradient elution) was used with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Was purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization. These steps are illustrated in the schematic diagram below.

[0282]

[Chemical 19] Example 25 GLP- at the ε-amino group of the added C-terminal lysine residue
Modification of 1. GLP-1 (1-36) -Lys ³⁷ (Nε-AEEA-AEEA-M
PA) -NH _2. 5TFA; His-Asp-Glu-Phe-Glu-Arg
-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp
-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala
-Ala-Lys-Glu-Phe-le-Ala-Trp-Leu-Val
-Lys-Gly-Arg-Lys (Nε-AEEA-AEEA-MPA) -N
H ₂ . Preparation of 5TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fm
oc-Trp-OH, Fmoc-Ala-OH, Fmoc-le-OH, Fm
oc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fm
oc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (
OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Pbf) -O.
H, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmo
c-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (N
-Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (O
tBu) -OH, Fmoc-Phe-OH, Fmoc-Glu (OtBu) -O
H, Fmoc-Asp (OtBu) -OH, Boc-His (N-Trt) -O
H (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of two AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimido prepionic acid (MPA) (step 3).
. Resin cleavage and product isolation was carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Ra
InIN) binary HPLC system for preparing _{(: 30~55% B (H 2} O 0.045% in TFA (A) and CH ₃ CN 0.045% in TFA (B)
) Gradient elution) of Phenomenex Luna 10 μ, phenyl-hexyl, 21 mm × 25 cm column and UV detector (Varian Dy).
Purified by preparative reverse-phase HPLC using namax UVD II) (λ 214 and 254 nm) at 9.5 mL / min for 180 min. The product is
Hewlett Packard LCMS-1 with geode array detector
RP-H with 100 series spectrometer and with electrospray ionization
Having> 95% purity as determined by PLC mass spectrometer, ESI-MS m
/ Z: C ₁₇₄ H ₂₆₅ N ₄₄ O ₅₆ (MH ⁺ ), calculated value 3868, measured value [M + H ₂ ] ₂ ⁺ 1
934, [M + H ₃ ] ³⁺ 1290, and [M + H ₄ ] ⁴⁺ 967. These steps are illustrated in the schematic diagram below.

[0284]

[Chemical 20] Example 26 GLP- at the ε-amino group of the added C-terminal lysine residue
Modification of 1. ^{GLP-1 (7-36) -Lys 37} (Nε-MPA) -NH 2. 4T
FA; His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-A
sp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-A
la-Ala-Lys-Glu-Phe-le-Ala-Trp-Leu-V
al-Lys-Gly-Arg- Lys (Nε-MPA) -NH 2. Preparation of 4TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fm
oc-Trp-OH, Fmoc-Ala-OH, Fmoc-le-OH, Fm
oc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fm
oc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (
OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Pbf) -O.
H, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmo
c-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (N
-Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated due to the addition of 3-maleimido prepionic acid (MPA) (step 3). Resin cleavage and product isolation using 85% TFA / 5%
Performed with TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-
_{55% B (H 2 O 0.045} % in TFA (A) and CH ₃ 0.04 in CN
5% TFA (B)) gradient elution) was used with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Was purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization.

Example 27 GLP at the ε-amino acid group of the added C-terminal lysine residue
-1 modification. GLP-1 (7-36) -Lys ³⁷ (Nε-AEEA-AEEA-
MPA) -NH _2. 4TFA His-Ala-Glu-Gly-Thr-Ph
e-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gl
u-Gly-Gln-Ala-Ala-Lys-Glu-Phe-lle-Al
a-Trp-Leu-Val-Lys-Gly-Arg-Lys (Nε-AEE
^{A-AEEA-MPA) -NH 2} . Preparation of 4TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fm
oc-Trp-OH, Fmoc-Ala-OH, Fmoc-le-OH, Fm
oc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fm
oc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (
OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Pbf) -O.
H, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmo
c-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (N
-Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of two AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimido prepionic acid (MPA) (step 3).
. Resin cleavage and product isolation was carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Ra
InIN) binary HPLC system for preparing _{(: 30~55% B (H 2} O 0.045% in TFA (A) and CH ₃ CN 0.045% in TFA (B)
) Gradient elution) of Phenomenex Luna 10 μ, phenyl-hexyl, 21 mm × 25 cm column and UV detector (Varian Dy).
Purified by preparative reverse-phase HPLC using namax UVD II) (λ 214 and 254 nm) at 9.5 mL / min for 180 min. The product is
Hewlett Packard LCMS-1 with geode array detector
RP-H with 100 series spectrometer and with electrospray ionization
It had> 95% purity as determined by PLC mass spectrometer.

Example 28 D-Al at the ε-amino group of the added C-terminal lysine residue
modification of a ² GLP-1. D-Ala ² GLP-1 (7-36) -Lys ³⁷ (Nε-
MPA) -NH _2. 4TFA His-d-Ala-Glu-Gly-Thr-
Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-
Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-lle-
Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys (Nε-M
PA) -NHh _2. Preparation of 4TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fm
oc-Trp-OH, Fmoc-Ala-OH, Fmoc-le-OH, Fm
oc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fm
oc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (
OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Pbf) -O.
H, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmo
c-Glu (OtBu) -OH, Fmoc-d-Ala-OH, Boc-His
(N-Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated due to the addition of 3-maleimido prepionic acid (MPA) (step 3). Resin cleavage and product isolation using 85% TFA / 5%
Performed with TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-
_{55% B (H 2 O 0.045} % in TFA (A) and CH ₃ 0.04 in CN
5% TFA (B)) gradient elution) was used with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Was purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization. These steps are illustrated in the schematic diagram below.

[0290]

[Chemical 21] Example 29 D-Al at the ε-amino group of the added C-terminal lysine residue
modification of a ² GLP-1. D-Ala ² GLP-1 (7-36) -Lys ³⁷ (Nε-
_{AEEA-AEEA-MPA) -NH 2} . 4TFA His-D-Ala-Gl
u-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Se
r-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Gl
u-Phe-lle-Ala-Trp-Leu-Val-Lys-Gly-Ar
g-Lys (Nε-AEEA- AEEA-MPA) -NH 2. Preparation of 4TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Arg (Pbf) -OH, Fmoc-Gly-OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fm
oc-Trp-OH, Fmoc-Ala-OH, Fmoc-le-OH, Fm
oc-Phe-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys
(TBoc) -OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fm
oc-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-Glu (
OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (Pbf) -O.
H, Fmoc-Ser (tBu) -OH, Fmoc-Ser (tBu) -OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmo
c-Glu (OtBu) -OH, Fmoc-d-Ala-OH, Boc-His
(N-Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of two AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimido prepionic acid (MPA) (step 3).
. Resin cleavage and product isolation was carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Ra
InIN) binary HPLC system for preparing _{(: 30~55% B (H 2} O 0.045% in TFA (A) and CH ₃ CN 0.045% in TFA (B)
) Gradient elution) of Phenomenex Luna 10 μ, phenyl-hexyl, 21 mm × 25 cm column and UV detector (Varian Dy).
Purified by preparative reverse-phase HPLC using namax UVD II) (λ 214 and 254 nm) at 9.5 mL / min for 180 min. The product is
Hewlett Packard LCMS-1 with geode array detector
RP-H with 100 series spectrometer and with electrospray ionization
It had> 95% purity as determined by PLC mass spectrometer. These steps are illustrated in the schematic diagram below.

[0292]

[Chemical formula 22]   Example 30 Exedes in the ε-amino group of the added C-terminal lysine residue
-4 (1-39) modification. Exendin-4 (1-39) -Lys⁴⁰(Nε-
MPA) -NH₂His-Gly-Glu-Gly-Thr-Phe-Thr
-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu
-Glu-Ala-Val-Arg-Leu-Phe-le-Glu-Trp
-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly
-Ala-Pro-Pro-Pro-Ser-Lys (N [epsilon] -MPA) -NH ₂ ． Preparation of 5TFA)   The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
  Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro
-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly
-OH, Fmoc-Ser-OH, Fmoc-Ser (tBu) -OH, Fmo
c-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmo
c-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-
Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (OtBu) -OH
, Fmoc-le-OH, Fmoc-Phe-OH, Fmoc-Leu-OH
, Fmoc-Arg (Bpf) -OH, Fmoc-Val-OH, Fmoc-A
la-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Glu (OtB
u) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH,
Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, Fm
oc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (
OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH,
Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-G
ly-OH, Boc-His (Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated due to the addition of 3-maleimido prepionic acid (MPA) (step 3). Resin cleavage and product isolation using 85% TFA / 5%
Performed with TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-
_{55% B (H 2 O 0.045} % in TFA (A) and CH ₃ 0.04 in CN
5% TFA (B)) gradient elution) was used with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Was purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization. These steps are illustrated in the schematic diagram below.

[0294]

[Chemical formula 23] Example 31 Modification of exendin-4 (1-39) at the ε-amino group of the added C-terminal lysine residue. Exedin 4 (1-39) -Lys ⁴⁰ (Nε-A
_{EEA-AEEA-MPA) -NH 2} . 5TFA; His-Gly-Glu-G
ly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-G
ln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-P
he-le-Glu-Trp-Leu-Lys-Asn-Gly-Gly-P
ro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-L
ys (Nε-AEEA-AEEA- MPA) -NH 2. The following protected amino acids were linked to the Link Amide using 5TFA automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro
-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala
-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser
(TBu) -OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmo
c-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (B
oc) -OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-
Glu (OtBu) -OH, Fmoc-le-OH, Fmoc-Phe-OH
, Fmoc-Leu-OH, Fmoc-Arg (Bpf) -OH, Fmoc-V
al-OH, Fmoc-Ala-OH, Fmoc-Glu (OtBu) -OH,
Fmoc-Glu (OtBu) -OH, Fmoc-Glu (OtBu) -OH,
Fmoc-Met-OH, Fmoc-Gln (Trt) -OH, Fmoc-Ly
s (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Leu-
OH, Fmoc-Asp (OtBu) -OH, Fmoc-Ser (tBu) -O
H, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-
Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtB
u) -OH, Fmoc-Gly-OH, Boc-His (Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of two AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimido prepionic acid (MPA) (step 3).
. Resin cleavage and product isolation was carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Ra
InIN) binary HPLC system for preparing _{(: 30~55% B (H 2} O 0.045% in TFA (A) and CH ₃ CN 0.045% in TFA (B)
) Gradient elution) of Phenomenex Luna 10 μ, phenyl-hexyl, 21 mm × 25 cm column and UV detector (Varian Dy).
Purified by preparative reverse-phase HPLC using namax UVD II) (λ 214 and 254 nm) at 9.5 mL / min for 180 min. The product is
Hewlett Packard LCMS-1 with geode array detector
RP-H with 100 series spectrometer and with electrospray ionization
It had> 95% purity as determined by PLC mass spectrometer. These steps are illustrated in the schematic diagram below.

[0296]

[Chemical formula 24] Example 32 Modification of Exendin-3 (1-39) at the ε-amino group of the added C-terminal lysine residue. Exendin-3 (1-39) -Lys ⁴⁰ (Nε
-MPA) -NH _2. 5TFA; His-Ser-Asp-Gly-Thr-P
he-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-G
lu-Glu-Glu-Ala-Val-Arg-Leu-Phe-le-G
lu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-S
er-Gly-Ala-Pro-Pro-Pro-Ser-Lys (Nε-MP
A) -NH _2. Preparation of 5TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro
-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly
-OH, Fmoc-Ser-OH, Fmoc-Ser (tBu) -OH, Fmo
c-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmo
c-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-
Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (OtBu) -OH
, Fmoc-le-OH, Fmoc-Phe-OH, Fmoc-Leu-OH
, Fmoc-Arg (Bpf) -OH, Fmoc-Val-OH, Fmoc-A
la-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Glu (OtB
u) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH,
Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, Fm
oc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (
OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH,
Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-S
er (OtBu) -OH, Boc-His (Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated due to the addition of 3-maleimido prepionic acid (MPA) (step 3). Resin cleavage and product isolation using 85% TFA / 5%
Performed with TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-
_{55% B (H 2 O 0.045} % in TFA (A) and CH ₃ 0.04 in CN
5% TFA (B)) gradient elution) was used with Phenomenex Luna.
10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Was purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization. These steps are illustrated in the schematic diagram below.

[0298]

[Chemical 25] Example 33 Modification of exendin-3 (1-39) at the ε-amino group of the added C-terminal lysine residue. Exendin-3 (1-39) -Lys ⁴⁰ (Nε
_{-AEEA-AEEA-MPA) -NH 2} . 5TFA; His-Ser-Asp
-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys
-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu
-Phe-lle-Glu-Trp-Leu-Lys-Asn-Gly-Gly
-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser
-Lys (Nε-AEEA-AEEA- MPA) NH 2. Preparation of 5TFA) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro
-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly
-OH, Fmoc-Ser-OH, Fmoc-Ser (tBu) -OH, Fmo
c-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmo
c-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-
Leu-OH, Fmoc-Trp-OH, Fmoc-Glu (OtBu) -OH
, Fmoc-le-OH, Fmoc-Phe-OH, Fmoc-Leu-OH
, Fmoc-Arg (Bpf) -OH, Fmoc-Val-OH, Fmoc-A
la-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Glu (OtB
u) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Met-OH,
Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, Fm
oc-Ser (tBu) -OH, Fmoc-Leu-OH, Fmoc-Asp (
OtBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (t
Bu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) OH, F
moc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Se
r (OtBu) -OH, Boc-His (Trt) -OH (step 1).

Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CH
3 equivalents of Pd (P) dissolved in Cl ₃ : NMM: HOAc (18: 1: 0.5)
This was achieved by treating the resin with a solution of Ph ₃ ) ₄ for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6
X 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL).
The synthesis was then re-automated for the addition of two AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimido prepionic acid (MPA) (step 3).
. Resin cleavage and product isolation was carried out with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Ra
InIN) binary HPLC system for preparing _{(: 30~55% B (H 2} O 0.045% in TFA (A) and CH ₃ CN 0.045% in TFA (B)
) Gradient elution) of Phenomenex Luna 10 μ, phenyl-hexyl, 21 mm × 25 cm column and UV detector (Varian Dy).
Purified by preparative reverse-phase HPLC using namax UVD II) (λ 214 and 254 nm) at 9.5 mL / min for 180 min. The product is
Hewlett Packard LCMS-1 with geode array detector
RP-H with 100 series spectrometer and with electrospray ionization
It had> 95% purity as determined by PLC mass spectrometer.

Example 34 Modification of HIV-1 DP 178 at the C-terminus. Modified HIV-1 DP 178 antifusogenic peptide Tyr-Thr-Ser-Le
u-le-His-Ser-Leu-le-Glu-Glu-Ser-Gl
n-Asn-Glu-Glu-Glu-Lys-Asn-Glu-Glu-Gl
u-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Se
r-Leu-Trp-Asn-Trp-Phe-Lys- (Nε-MPA) -N
Preparation of H ₂ ) The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Mtt) -OH, Fmo.
c-Phe-OH, Fmoc-Trp (Boc) -OH, Fmoc-Asn (T
rt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Leu-OH,
Fmoc-Ser (tBu) -OH, Fmoc-Ala-OH, Fmoc-Tr
p (Boc) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (
tBu) -OH, Fmoc-Leu-OH, Fmoc-Glu (tBu) -OH
, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu (Tb
u) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu (tBu)
-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -O
H, Fmoc-Glu (tBu) -OH, Fmoc-Gln (Trt) -OH,
Fmoc-Gln (Trt) -OH, Fmoc-Asn (Trt) -OH, Fm
oc-Gln (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc
-Glu (tBu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-1
le-OH, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, F
moc-His (Trt) -OH, Fmoc-le-OH, Fmoc-Leu
-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -O
H and Boc-Tyr (tBu) -OH. Manual synthesis is used for the remaining steps: selective removal of Mtt group and HBTU / HOBt / DIEA in DMF.
Coupling of maleimidopropionic acid (MPA) with activity. The target molecule
Removed from resin; the product is isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid on lyophilization.

Example 35 Modification of HIV-1 DP 107 at the C-terminus. Modified HIV-1 DP 107 antifusogenic peptide Asn-Asn-Leu-Le.
u-Arg-Ala-lle-Glu-Ala-Glu-Glu-His-Le
u-Leu-Glu-Leu-Thr-Val-Trp-Glu-le-Ly
s-Glu-Leu-Glu-Ala-Arg-le-Leu-Ala-Va
l-Glu-Arg-Tyr-Leu-Lys-Asp-Glu-Lys- (N
Preparation of ε-MPA) -NH ₂ ) The following protected amino acids were converted to Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Mtt) -OH, Fmo.
c-Gln (Trt) -OH, Fmoc-Asp (tBu) -OH, Fmoc-
Lys (Boc) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (tBu)-
OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Leu-
OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf) -OH, Fmoc
-Ala-OH, Fmoc-Gln (Trt) -OH, Fmoc-Leu-OH
, Fmoc-Gln (Trt) -OH, Fmoc-Lys (Boc) -OH, F
moc-le-OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp
(Boc) -OH, Fmoc-Val-OH, Fmoc-Thr (tBu) -O
H, Fmoc-Leu-OH, Fmoc-Gln (Trt) -OH, Fmoc-
Leu-OH, Fmoc-Leu-OH, Fmoc-His (Trt) -OH,
Fmoc-Gln (Trt) -OH, Fmoc-Gln (Trt) -OH, Fm
oc-Ala-OH, Fmoc-Glu (tBu) -OH, Fmoc-lle-
OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc
-Leu-OH, Fmoc-Leu-OH, Fmoc-Asn (Trt) -OH
, Fmoc-Asn (Trt) -OH. Manual synthesis is used for the remaining steps: selective removal of the Mtt group and coupling of maleimidopropionic acid (MPA) with HBTU / HOBt / DIEA activity in DMF. Target analog
Removed from resin; the product is isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid on lyophilization.

(2. Modification of therapeutic peptide at N-terminus) (Example 36) Modification of RSV peptide at ε-amino group of added N-terminal lysine residue (Nε-MPA) -Lys-Val-lle- Thr-lle-
Glu-Leu-Ser-Asn-le-Lys-Glu-Asn-Lys-
Met-Asn-Gly-Ala-Lys-Val-Lys-Leu-lle-
Lys-Gln-Glu-Leu-Asp-Lys-Tyr-Lys-Asn-
Preparation of Ala-Val) Solid phase peptide synthesis of modified RSV peptide on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synthesizer.
And Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Val-OH, Fmoc.
-Ala-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Lys (Boc)
-OH, Fmoc-Asp (tBu) -OH, Fmoc-Leu-OH, Fmo
c-Glu (tBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-
Lys (Boc) -OH, Fmoc-le-OH, Fmoc-Leu-OH,
Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Ly
s (Boc) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fm
oc-Asn (Trt) -OH, Fmoc-Met-OH, Fmoc-Lys (
Boc) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Glu (tB
u) -OH, Fmoc-Lys (Boc) -OH, Fmoc-le-OH, F
moc-Asn (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmo
c-Leu-OH, Fmoc-Glu (tBu) -OH, Fmoc-le-O
H, Fmoc-Thr (tBu) -OH, Fmoc-le-OH, Fmoc-
Val-OH, Fmoc-Lys (Aloc) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU) And diisopropylethylamine (DIEA
) To activate. Removal of the Fmoc protecting group is accomplished in 20 minutes with a solution of 20% (V / V) piperidine in N, N-dimethylformamide (DMF) (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 2 with a solution of 3 equivalents of Pd (PPh3) 4 dissolved in 5 mL of CHCl3: NMM: HOAc (18: 1: 0.5). Achieved by treating the resin for hours (
Step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% in DCM.
HOAc (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL)
) Wash with. The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et2O (step 4). The product was converted into Varian (Rai
nin) Preparative binary HPLC system (: 30-55% B (0 in H ₂ O)
． 045% TFA (A) and 0.045% TFA (B) in CH ₃ CN)
Gradient elution) of Phenomenex Luna 10 μ Phenyl-
Hexyl, 21 mm x 25 cm column and UV detector (Varian Dyn
amax UVD II) (λ214 and 254 nm) and purified by preparative reverse phase HPLC at 9.5 mL / min for 180 min, RP-HPLC.
The desired DAC is obtained in> 95% purity as determined by.

Example 37 Modification of neuropeptide Y at the ε-amino group of the added N-terminal lysine residue. (N-εMPA) -Lys-Tyr-Pro-Ser-L
ys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-P
ro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-S
Preparation of er-Ala-Leu) Solid phase peptide synthesis of modified neuropeptide Y on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synthesi.
Zer and Fmoc protected Rink Amide MBHA.
The following protected amino acids are sequentially added to the resin: Fmoc-Leu-OH, F
moc-Ala-OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr
(TBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Arg (P
bf) -OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-
Asp (tBu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Al
a-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-As
p (tBu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Gly-
OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc
-Glu (tBu) -OH, Fmoc-Pro-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, F
moc-Tyr (tBu) -OH, Fmoc-Lys (Aloc) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU) And activated with diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is accomplished by N, N-
Achieve in 20 minutes using a solution of 20% (V / V) piperidine in dimethylformamide (DMF) (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl3: NMM: HOAc (18: 1: 0.5
This is achieved by treating the resin with a solution of 3 equivalents of Pd (PPh3) 4 dissolved in 2) for 2 hours (step 2). The resin was then treated with CHCl ₃ (6 × 5 mL)
Wash with 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). During every coupling,
The resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide was added to 85% TFA / 5% TIS / 5.
Cleavage from the resin with% thioanisole and 5% phenol, followed by:
Precipitate with Et2O cooled with dry ice (step 4). The product is
arian (Rainin) Preparative Binary HPLC System (: 30-55
% B (0.045% TFA (A) in H ₂ O and 0.045% in CH ₃ CN
Gradient elution of TFA (B)) using Phenomenex Luna 1
0 μ Phenyl-hexyl, 21 mm × 25 cm column and UV detector (Va
Rian Dynamax UVD II) (λ214 and 254 nm) was used to purify by preparative reverse phase HPLC for 180 min at 9.5 mL / min and the desired DAC in> 95% purity as determined by RP-HPLC. To get

Example 38 Modification of neuropeptide Y at the ε-amino group of the added N-terminal lysine residue. (N-εMPA) -Lys-Tyr-Pro-Ser-L
ys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-P
ro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-S
Preparation of er-Ala-Leu) Solid phase peptide synthesis of modified neuropeptide Y on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synthesi.
Run with zer and Fmoc protected Rink Amide MBHA.
The following protected amino acids are sequentially added to the resin: Fmoc-Leu-OH, F
moc-Ala-OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr
(TBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Arg (P
bf) -OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-
Asp (tBu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Al
a-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-As
p (tBu) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Gly-
OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc
-Asp (tBu) -OH, Fmoc-Pro-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, F
moc-Tyr (tBu) -OH, Fmoc-Lys (Aloc) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (HBTU) And activated with diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is accomplished by N, N-
Achieve in 20 minutes using a solution of 20% (V / V) piperidine in dimethylformamide (DMF) (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl3: NMM: HOAc (18: 1: 0.5
This is achieved by treating the resin with a solution of 3 equivalents of Pd (PPh3) 4 dissolved in 2) for 2 hours (step 2). The resin was then treated with CHCl ₃ (6 × 5 mL)
Wash with 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). During every coupling,
The resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide was added to 85% TFA / 5% TIS / 5.
Cleavage from the resin with% thioanisole and 5% phenol, followed by:
Precipitate with Et2O cooled with dry ice (step 4). The product is
arian (Rainin) Preparative Binary HPLC System (: 30-55
% B (0.045% TFA (A) in H ₂ O and 0.045% in CH ₃ CN
Gradient elution of TFA (B)) using Phenomenex Luna 1
0 μ Phenyl-hexyl, 21 mm × 25 cm column and UV detector (Va
Rian Dynamax UVD II) (λ214 and 254 nm) was used to purify by preparative reverse phase HPLC for 180 min at 9.5 mL / min and the desired DAC in> 95% purity as determined by RP-HPLC. To get

Example 39 Dyn A at epsilon amino group of added N-terminal lysine residue
Modification of 1-13- (Nε-MPA) -Dyn A 1-13-NH ₂ (Nε-
MPA) -Lys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg.
-Ile-Arg-Pro-Lys-Leu Synthesis) Solid phase peptide synthesis of the modified Dyn A 1-13 analog on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synt.
Run with hesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Lys (
Boc) -OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH
, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-1
le-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Arg (Pbf)
-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly
-OH, Fmoc-Gly-OH, Fmoc-Tyr (tBu) -OH, Fmo
c-Lys (Aloc) -OH. They were treated with N, N-dimethylformamide (D
MF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate (
Activation with HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by adding 20 in N, N-dimethylformamide (DMF).
% (V / V) piperidine solution in 20 minutes. Lys (Aloc
) Selective deprotection of the group is performed manually and 5 mL of CHCl3: NMM: H
This is accomplished by treating the resin with a solution of 3 equivalents of Pd (PPh3) 4 dissolved in OAc (18: 1: 0.5) for 2 hours (step 2). Then, this resin
CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL), DC
Wash with M (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). The resin was washed with N, N-dimethylformamide (DMF) between all couplings.
Wash twice and wash three times with isopropanol. 85% of this peptide
Cleavage from the resin with TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et2O cooled with dry ice (step 4). The product is a binary HPL for Varian (Rainin) preparation.
C System (: gradient elution of _{30~55% B (H 2 0.045%} TFA (A in O) and CH ₃ CN 0.045% in TFA (B))) using, Phen
Omenex Luna 10μ Phenyl-hexyl, 21mm x 25cm column and UV detector (Varian Dynamax UVD II) (λ2
14 and 254 nm) for> 180 min at 9.5 mL / min by preparative reverse phase HPLC and> 95 as determined by RP-HPLC.
The desired DAC is obtained in% purity.

Example 40 Modification of Dyn A 2-17-NH ₂ in N-Terminal Glycine-MPA-AEA ₃ -Dyn A 2-17-NH ₂ (MPA-AEA-AEA
-AEA) -Gly-Gly-Phe-Leu-Arg-Arg-lle-Ar
Synthesis of g-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Glu) The following protected amino acids and maleimides were bound to Rin using automated peptide synthesis.
k Amide MBHA resin was continuously added: Fmoc-Gln (Tr
t) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Asp (OtBu
) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Lys (Boc)-
OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc
-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-le-OH
, Fmoc-Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, F
moc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, F
moc-Gly-OH, Fmoc-AEA-OH, Fmoc-AEA-OH, F
moc-AEA-OH and MPA. The target dynorphin analog is then
Removed from resin; product isolated by precipitation and purified by preparative HPLC to give the desired product on lyophilization in 32% yield as a pale yellow solid. Analytical HPLC showed the product to be> 95% pure at Rt = 33.44 min. ESI-MS m / z: C 109 H 172 N 35 O 29 (MH +), calcd 24
36.8, found MH3 ⁺ 813.6.

[0307]

[Chemical formula 26] Example 42 Modification of kringle-5 at the ε-amino group of the added N-terminal lysine residue. (Nε-MPA) -Lys-Pro-Arg-Lys-Leu-
_{Tyr-Asp-Tyr-NH 2} . 2TFA Preparation) Solid phase peptide synthesis of modified kringle-5 analogs on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Syntheses.
Run with izer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Tyr (tBu
) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Tyr (tBu)-
OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc
-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Lys (Al
oc) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ',
Activation with N'-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is accomplished in 20 minutes with a solution of 20% (V / V) piperidine in N, N-dimethylformamide (DMF) (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl3: NMM: HOAc
This is accomplished by treating the resin with a solution of 3 equivalents of Pd (PPh3) 4 in (18: 1: 0.5) for 2 hours (step 2). Then, this resin is treated with CHC
_{l 3 (6 × 5mL),} 20% HOAc (6 × 5mL) in DCM, DCM (6
X 5 mL), and DMF (6 x 5 mL). This synthesis is then
Re-automating for the addition of maleimido prepionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. This peptide was added to 85% TFA
Cleave from the resin with / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice-cooled Et2O (step 4). The product was loaded onto a Varian (Rainin) preparative binary HPLC system (: 30-55% B (0.045% TFA (A) and CH ₃ C in H ₂ O).
Gradient elution of 0.045% TFA (B)) in N).
180 by preparative reverse phase HPLC at 9.5 mL / min using ex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) (λ214 and 254 nm).
Purify over minutes to obtain the desired DAC in> 95% purity as determined by RP-HPLC.

Example 43 Modification of Kringle-5 at the N-Terminal Proline (MPA-A
EEA) -Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys.
-NH _2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH (
Step 1). Deprotection of the terminal Fmoc group is achieved with 20% piperidine (step 2), followed by coupling of 3-MPA. The resulting Fmoc-AEEA
-Deprotection of the peptide with 20% piperidine in DMF followed by 3-M
Allow the addition of PA (step 3). Resin cleavage and product isolation was performed with 86% T
It performed using FA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold Et ₂ O (Step 4). The product was loaded onto a Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax).
Varian (Rain using a Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column with UVD II) (λ214 and 254 nm).
in) Purify by preparative reverse phase HPLC using a preparative binary HPLC system. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization.

[0309]   (Example 44 Modification of kringle-5 in N-terminal proline. (MPA)-
Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH₂．
Preparation of 2TFA)   The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH (
Step 1). Deprotection of the terminal Fmoc group was achieved with 20% piperidine (engineering
Step 2), followed by coupling of 3-MPA (step 3). Resin cutting and
Product isolation from 86% TFA / 5% TIS / 5% H₂O / 2% Thioani
Performed with sole and 2% phenol, followed by dry ice cooled Et ₂ Precipitate with O (step 4). The product was treated with Dynamax C₁₈, 60Å,
8 μm guard module, 21 mm x 25 cm column and UV detector (Va
Rian Dynamax UVD II) (λ214 and 254 nm)
Eta Dynamax C₁₈, 60Å, 8μm, 21mm × 25cm column
Prepared using a Varian (Rainin) preparative binary HPLC system
Purify by preparative reverse phase HPLC. The product is H with a geode array detector.
Using an Hewlett Packard LCMS-1100 series spectrometer and
Determined by RP-HPLC mass spectrometer with electrospray ionization
> 95% pure.

Example 45 Modification of kringle-5 in N-terminal tyrosine (MPA-A
EEA) -Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu.
_{-Tyr-Asp-Tyr-NH 2} . Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH,
Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fm
oc-Thr (tBu) -OH, Fmoc-Tyr (tBu) OH (step 1).
Deprotection of the terminal Fmoc group is achieved with 20% piperidine (step 2), followed by Fmoc-AEEA coupling. Fmoc-AEEA-
Deprotection of the peptide with 20% piperidine in DMF was followed by 3-MP
Allows addition of A (step 3). Resin cleavage and product isolation using 86% TF
It performed using A / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold Et ₂ O (Step 4). The product was applied to Dynamax C ₁₈ , 60Å, 8 μm guard module,
21 mm x 25 cm column and UV detector (Varian Dynamax
Dynamax C ₁₈ with UVD II) (λ 214 and 254 nm),
Varian (Raini using a 60Å, 8 μm, 21 mm × 25 cm column)
n) Purify by preparative reverse phase HPLC using a preparative binary HPLC system. The product was a Hewlett Packard equipped with a geode array detector.
d had> 95% purity as determined by RP-HPLC mass spectrometer using LCMS-1100 series spectrometer and using electrospray ionization.

[0311]   (Example 46 Modification of kringle-5 in N-terminal tyrosine. (MPA)-
Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu-Tyr-
Asp-Tyr-NH₂． Preparation of 2TFA)   The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH,
Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fm
oc-Thr (tBu) -OH, Fmoc-Tyr (tBu) -OH (step 1)
. Deprotection of the terminal Fmoc group was achieved with 20% piperidine (step 2),
Then, Fmoc-AEEA is coupled (step 3). Resin cutting and
Product isolation from 86% TFA / 5% TIS / 5% H₂O / 2% Thioani
Performed with sole and 2% phenol, followed by dry ice cooled Et ₂ Precipitate with O (step 4). The product was treated with Dynamax C₁₈, 60Å,
8 μm guard module, 21 mm x 25 cm column and UV detector (Va
Rian Dynamax UVD II) (λ214 and 254 nm)
Eta Dynamax C₁₈, 60Å, 8μm, 21mm × 25cm column
Prepared using a Varian (Rainin) preparative binary HPLC system
Purify by preparative reverse phase HPLC. The product is H with a geode array detector.
Using an Hewlett Packard LCMS-1100 series spectrometer and
Determined by RP-HPLC mass spectrometer with electrospray ionization
> 95% pure.

Example 47 Modification of kringle-5 at the N-terminal arginine (MPA-
AEEA) -Arg-Asn-Pro-Asp-Gly-Asp-Gly-Pr.
o-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg-Ly
s-Leu-Tyr-Asp- Tyr-NH 2. Preparation of 3TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide:
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH,
Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fm
oc-Thr (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc
-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc
-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc
-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asp (O
tBu) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH
, Fmoc-Arg (Pbf) -OH (step 1). Deprotection of the terminal Fmoc group,
Achieved with 20% piperidine (step 2), followed by coupling of 3-MPA. Deprotection of the resulting Fmoc-AEEA-peptide with 20% piperidine in DMF allows subsequent addition of 3-MPA (step 3). Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5% H ₂ O / 2.
Performed with% thioanisole and 2% phenol, followed by precipitation with dry ice cold Et ₂ O (step 4). The _{product, Dynamax C 1 8, 60Å,} 8μm guard module, 21 mm × 25 cm column and UV
Detector (Varian Dynamax UVD II) (λ 214 and 25
Dynamax C ₁₈ , 60 Å, 8 μm, 21 mm x 25 cm with 4 nm)
Purify by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system with a column. The product had> 95% purity as determined using a Hewlett Packard LCMS-1100 series spectrometer with Geode array detector and by RP-HPLC mass spectrometer with electrospray ionization.

Example 48 Modification of kringle-5 at the N-terminal arginine (MPA).
-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly
-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg
-Lys-Leu-Tyr-Asp- Tyr-NH 2. Preparation of 3TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide:
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH,
Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fm
oc-Thr (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc
-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc
-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc
-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asp (O
tBu) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH
, Fmoc-Arg (Pbf) -OH (step 1). Deprotection of the terminal Fmoc group,
Achieved with 20% piperidine (step 2), followed by coupling of Fmoc-AEEA (step 3). Resin cleavage and product isolation using 86% TFA
/ 5% TIS / 5% with H ₂ O / 2% thioanisole and 2% phenol executed, followed by precipitation by dry-ice cold Et ₂ O (Step 4
). The product was applied to Dynamax C ₁₈ , 60Å, 8 μm guard module, 2
1 mm x 25 cm column and UV detector (Varian Dynamax U
Dynamax C ₁₈ , 6 with VD II) (λ 214 and 254 nm)
Varian (Rainin using 0Å, 8 μm, 21 mm × 25 cm column)
) Purify by preparative reverse phase HPLC using a preparative binary HPLC system. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an LCMS-1100 series spectrometer and using electrospray ionization.

[0314]   (Example 49 Modification of kringle-5 in N-terminal arginine. (MPA-
AEEA) -Arg-Asn-Pro-Asp-Gly-Asp-Val-Gl
y-Gly-Pro-Trp-NH₂． Preparation of TFA)   The following protected amino acids were linked to Rink Amide using automated peptide synthesis.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fm
oc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)
-OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (
Pbf) -OH (step 1). Deprotection of the terminal Fmoc group, 20% piperidine
Is achieved (step 2), followed by Fmoc-AEEA coupling.
Use 20% piperidine in DMF of the resulting Fmoc-AEEA-peptide
Deprotection allows subsequent addition of 3-MPA (step 3). Resin cutting and
Product isolation from 86% TFA / 5% TIS / 5% H₂O / 2% Thioani
Performed with sole and 2% phenol, followed by dry ice cooled Et ₂ Precipitate with O (step 4). The product was treated with Dynamax C₁₈, 60Å,
8 μm guard module, 21 mm x 25 cm column and UV detector (Va
Rian Dynamax UVD II) (λ214 and 254 nm)
Eta Dynamax C₁₈, 60Å, 8μm, 21mm × 25cm column
Prepared using a Varian (Rainin) preparative binary HPLC system
Purify by preparative reverse phase HPLC. The product is H with a geode array detector.
Using an Hewlett Packard LCMS-1100 series spectrometer and
Determined by RP-HPLC mass spectrometer with electrospray ionization
> 95% pure.

Example 50 Modification of kringle-5 at the N-terminal arginine (MPA).
-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gly
-Pro-Trp-NH _2. Preparation of TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide:
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fm
oc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)
-OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (
Pbf) -OH (step 1). Deprotection of the terminal Fmoc group is achieved with 20% piperidine (step 2), followed by coupling of 3-MPA (step 3).
. Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5% H ₂ O.
/ Run with 2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold Et ₂ O (Step 4). The product was
C _18, Dynamax C ₁₈ with 60 Å, 8 [mu] m guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax UVD II) (λ214 and 254nm), 60Å, 8μm, 21mm × 25
Purify by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system using a cm column. The product had> 95% purity as determined using a Hewlett Packard LCMS-1100 series spectrometer with Geode array detector and by RP-HPLC mass spectrometer with electrospray ionization.

Example 51 Modification of kringle-5 at the N-terminal arginine (MPA-
AEEA) -Arg-Lys-Leu- Tyr-Asp-Tyr-NH 2. 2T
Preparation of FA) The following protected amino acids were linked to Rink Amide using an automated peptide synthesis.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH (step 1). Deprotection of the terminal Fmoc group was achieved with 20% piperidine (step 2), followed by Fmoc
-Coupling of AEEA. DM of the resulting Fmoc-AEEA-peptide
Deprotection with 20% piperidine in F allows subsequent addition of 3-MPA (step 3). Resin cleavage and product isolation was performed using 86% TFA / 5% TI.
Performed with S / 5% H ₂ O / 2% thioanisole and 2% phenol, followed by precipitation with dry ice cold Et ₂ O (step 4). The product was applied to Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25.
cm column and UV detector (Varian Dynamax UVD II)
Dynamax C ₁₈ , 60Å, 8 μm with (λ 214 and 254 nm)
, Purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system using a 21 mm x 25 cm column. The product was a Hewlett Packard LCMS- equipped with a Geode array detector.
RP- with 1100 series spectrometer and with electrospray ionization
It had> 95% purity as determined by HPLC mass spectroscopy.

Example 52 Modification of kringle-5 at the N-terminal arginine (MPA).
-Arg-Lys-Leu-Tyr- Asp-Tyr-NH 2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Tyr (tBu) -OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) -OH, Fmoc-Leu-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH (step 1). Deprotection of the terminal Fmoc group was achieved with 20% piperidine (step 2), followed by 3-MP.
Coupling A (step 3). Resin cleavage and product isolation was performed with 86% T
It performed using FA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold Et ₂ O (Step 4). The product was loaded onto a Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax).
Varian (Rain using a Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column with UVD II) (λ214 and 254 nm).
in) Purify by preparative reverse phase HPLC using a preparative binary HPLC system. The product was a Hewlett Packard equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an rd LCMS-1100 series spectrometer and using electrospray ionization.

Example 53 Modification of Kringle-5 at N-terminal Proline. (MPA-A
EEA) -Pro-Arg-Lys- Leu-Tyr-Asp-NH 2. 2TF
Preparation of A) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) -OH, Fmo
c-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (P
bf) -OH, Fmoc-Pro-OH (step 1). Deprotection of the terminal Fmoc group was achieved with 20% piperidine (step 2), followed by Fmoc-AEEA.
Coupling. 20 of the resulting Fmoc-AEEA-peptide in DMF
Deprotection with% piperidine allows subsequent addition of 3-MPA (step 3). Resin cleavage and product isolation, 86% TFA / 5% TIS / 5%
Performed with H ₂ O / 2% thioanisole and 2% phenol, followed by precipitation with dry ice cold Et ₂ O (step 4). The product is
max C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax UVD II) (λ214
Dynamax C ₁₈ , 60 Å, 8 μm, 21 mm
Binary HP for Varian (Rainin) preparation using x25 cm column
Purify by preparative reverse phase HPLC using an LC system. The product had> 95% purity as determined using a Hewlett Packard LCMS-1100 series spectrometer with Geode array detector and by RP-HPLC mass spectrometer with electrospray ionization.

Example 54 Modification of Kringle-5 at N-terminal Proline. (MPA)-
Pro-Arg-Lys-Leu- Tyr-Asp-NH 2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were linked to Rink Amide.
Add continuously to MBHA resin: Fmoc-Lys (Boc) -OH, Fm
oc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) -OH, Fmo
c-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (P
bf) -OH, Fmoc-Pro-OH (step 1). Deprotection of the terminal Fmoc group is achieved with 20% piperidine (step 2), followed by coupling of 3-MPA (step 3). Resin cleavage and product isolation, 86% TFA / 5%
It performed using _{TIS / 5% H 2 O /} 2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold Et ₂ O (Step 4). The product was applied to Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm.
X 25 cm column and UV detector (Varian Dynamax UVD
II) Dynamax C ₁₈ , 60Å with (λ214 and 254 nm),
Purify by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system using an 8 μm, 21 mm × 25 cm column. The product was a Hewlett Packard LC equipped with a geode array detector.
It had a> 95% purity as determined by an RP-HPLC mass spectrometer using an MS-1100 series spectrometer and using electrospray ionization.

3. Modifications at Internal Amino Acids Example 55 Synthesis of Lys ²⁶ (ε-MPA) GLP-1 (7-36) -NH _{2 Of} modified GLP-1 analogs on a 100 μmol scale. Solid-phase peptide synthesis was performed manually and using Fmoc-protected Rink Amide MBHA resin.
Run on ymphony Peptide Synthesizer. The following protected amino acids were sequentially added to the resin: Fmoc-Arg (Pbf)-
OH, Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc
-Val-OH, Fmoc-Leu-OH, Fmoc-Trp (Boc) -OH
, Fmoc-Ala-OH, Fmoc-lle-OH, Fmoc-Phe-OH
, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (Aloc) -OH
, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln (Tr
t) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH,
Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Se
r (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-
OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH
, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-T
hr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu
) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uronium hexafluorophosphate (HBTU). ) And diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was reduced to 20% (V
/ V) piperidine in N, N-dimethylformamide (DMF) solution 20
Activate for minutes (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0.5). This is achieved by treating the resin for 2 hours at (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), DCM
20% HOAc in (6 x 5 mL), DCM (6 x 5 mL), and DMF (
6 × 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 3). 86% for resin cleavage and product isolation
TFA / 5% TIS / 5% with H ₂ O / 2% thioanisole and 2% phenol executed, followed by precipitation by cold Et ₂ O in dry ice (Step 4). The product was loaded onto a Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian Dyn.
Dynama with amax UVD II) (λ 214 and 254 nm)
x C ₁₈ , 60Å, 8 μm, Varian (using a 21 mm x 25 cm column
Rainin) Preparative Reversed-Phase HPLC Using a Preparative Binary HPLC System
Purified to give the desired DAC with> 95% purity as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

[0321]

[Chemical 27] D. Preparation of Modified Peptides from Peptides Containing One Free Cysteine The preparation of maleimide peptides from therapeutic peptides containing one free cysteine is exemplified by peptide synthesis as described below. . The peptide may be modified at the N-terminus, the C-terminus or at the amino acid located between the N- and C-termini.

Peptides Containing All Multiple Cysteine Residues with Multiple Protective Functional Groups and One Free Cysteine Residue (ie All Cysteine Residues Except One Cysteine Residue are Constrained as Disulfides) Preparation of Maleimide Peptides from. Ligation from internal amino acids in the native sequence as in Example 5. Free cysteine residues must be capped or replaced with another amino acid (eg, alanine, methionine, etc.).

If the peptide contains one cysteine, this cysteine must remain capped after addition of the maleimide. If cysteine is involved in the binding site, there is a great deal of lost potential and an assessment of how cysteine is capped by the protecting group must be made. If the cysteine remains capable, the synthetic route is similar to example (i) above. Examples of therapeutic peptides containing one cysteine are G _α (α subunit of G therapeutic peptide binding protein), rat brain carbon monoxide synthetase block peptide 724-739 fragment, α of human [Tyr0] inhibin. Subunits 1-32, HIV envelope protein 254-274 fragment and P34 cdc2 kinase fragment.

(1. Modification at N-Terminal) (Example 56) Modification of inhibin peptide in added N-terminal lysine.
(Nε-MPA) -Lys-Tyr-Ser-Thr-Pro-Leu-Met
-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu
-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu
-Pro-Ala-Ala-Ala-His-Ala-Asn-Cys-His
-Preparation of Arg) Solid phase peptide synthesis of modified inhibin peptide analogs on a 100 μmol scale was performed by manual solid phase synthesis, Symphony Peptide Synt.
Run with hesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids are sequentially added to the resin: Fmoc-Arg
(Pbf) -OH, Fmoc-His (Boc) -OH, Fmoc-Cys (T
rt) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Ala-OH,
Fmoc-His (Boc) -OH, Fmoc-Ala-OH, Fmoc-Al
a-OH, Fmoc-Pro-OH, Fmoc-Glu (tBu) -OH, Fm
oc-Glu (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro-
OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH
, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Arg (Pb
f) -OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-S
er (tBu) -OH, Fmoc-Pro-OH, Fmoc-Ser (tBu)
-OH, Fmoc-Trp (Boc) -OH, Fmoc-Pro-OH, Fmo
c-Trp (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-
Met-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-
Thr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ty
r (tBu) -OH, Fmoc-Lys (Aloc) -OH. Let them be N, N
-Dissolved in dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (D
Activate with IEA). Removal of the Fmoc protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and dissolved in 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0.5) 3.
Achieved by treating the resin with a solution of an equivalent amount of Pd (PPh ₃ ) ₄ for 2 hours (
Step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL), DCM (6 × 5 mL), and DMF (6 × 5 mL).
) Was washed with. The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). Between every coupling, the resin is washed 3 times with N, N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved from the resin with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 4). The product was converted into Varian (Rai
nin) Preparative binary HPLC system (: 30-55% B (0 in H ₂ O)
． 045% TFA (A) and 0.045% TFA (B) in CH ₃ CN)
Gradient elution) of Phenomenex Luna 10 μ Phenyl-
Hexyl, 21 mm x 25 cm column and UV detector (Varian Dyn
amax UVD II) (λ214 and 254 nm), purified by preparative reverse phase HPLC at 9.5 mL / min for 180 min, RP-HPLC.
The desired DAC is obtained in> 95% purity as determined by.

Example 57 Modification of RSV antifusogenic peptide at the N-terminus. 3- (MP
A) -Val-Ile-Thr-Ile-Glu-Leu-Ser-Asn-l
le-Lys-Glu-Asn-Lys-Cys-Asn-Glu-Ala-L
ys-Val-Lys-Leu-le-Lys-Glu-Glu-Leu-A
Preparation of sp-Lys-Tyr-Lys-Asn-Ala-Val) First, cysteine (Cys) was added to methionine (Met) in the native sequence.
Replaced with. Solid-phase peptide synthesis of modified anti-RSV analogs on a 100 μmol scale was performed on a Symphony Peptide Synthesizer using O-benzo in Fmoc-protected Rink Amide MBHA resin, Fmoc-protected amino acid, N, N-dimethylformamide (DMF). Triazole-1-
Ill-N, N, N ', N'-Tetramethyl-uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM) with activation and with piperidine deprotection of the Fmoc group. (Step 1). Terminal Fmo
Deprotection of the c group was achieved with 20% piperidine (step 2), followed by 3-
Coupling of MPA (step 3). 85% for resin cleavage and product isolation
Performed with TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product, Varian (Rainin) binary HPLC system for preparing _{(: 30~55% B (H 2} 0.045% TFA (A in O) and CH ₃ CN 0.045% in TFA in (B)) Gradient elution)
18 by preparative reverse phase HPLC at 9.5 mL / min using a Nex Luna 10 [mu] Phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) ([lambda] 214 and 254 nm).
Purify over 0 minutes to obtain the desired DAC in> 95% purity as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

[0326]

[Chemical 28]   (2. Modification at C-terminal)   Example 58 Modification of the inhibin peptide at the added C-terminal lysine.
(Nε-MPA) -Lys-Cys-Asn-Leu-Lys-Glu-Asp
-Gly-lle-Ser-Ala-Ala-Lys-Asp-Val-Lys
Preparation)   Solid phase peptide of modified inhibin peptide analog on a scale of 100 μmol
Peptide synthesis, manual solid phase synthesis, Symphony Peptide Synt
Hezizer and Fmoc protected Rink Amide MBHA
To go. The following protected amino acids are sequentially added to the resin: Fmoc-Lys
(Boc) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -O
H, Fmoc-Lys (Boc) -OH, Fmoc-Ala-OH, Fmoc-
Ala-OH, Fmoc-Ser (tBu) -OH, Fmoc-le-OH,
Fmoc-Gly-OH, Fmoc-Asp (tBu) -OH, Fmoc-Gl
u (tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Leu-
OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Trt) -OH
, Fmoc-Lys (Aloc) -OH. They are N, N-dimethylformua
Dissolve in amide (DMF) and according to the sequence O-benzotriazole-1
-Yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphine
(HBTU) and diisopropylethylamine (DIEA).
Sexualize. Removal of the Fmoc protecting group was accomplished by removing 20% (V / V) piperidine in N, N-di-
Activate with a methylformamide (DMF) solution for 20 minutes (step 1). L
Selective deprotection of the ys (Aloc) group was performed manually and 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0.5) dissolved in 3 equivalents of Pd (PPh₃ )_FourThis is achieved by treating the resin with the solution of 2 hours (step 2). Then
This resin is₃(6 × 5 mL), 20% HOAc in DCM (6 × 5 mL)
mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). Next
In, the synthesis is automated for the addition of 3-maleimidopropionic acid (step
3). During all couplings, the resin was loaded with N, N-dimethylformamide (D
Wash 3 times with MF) and 3 times with isopropanol. This peptide
, 85% TFA / 5% TIS / 5% thioanisole and 5% pheno
Resin and then dry ice-cooled Et.₂By O
Precipitate (step 4). The product is a Varian (Rainin) preparative binder.
Lee HPLC system (: 30-55% B (H₂0.045% TFA in O (
A) and CH₃Gradient elution of 0.045% TFA (B)) in CN)
, Phenomenex Luna 10μ Phenyl-hexyl, 21mm x
25 cm column and UV detector (Varian Dynamax UVD I
I) (λ 214 and 254 nm) using preparative reverse phase HPLC 9.5.
Purify for 180 min at mL / min, as determined by RP-HPLC
First, the desired DAC is obtained with> 95% purity.

Example 59 Modification of RSV fusogenic peptide at the C-terminus. Val-ll
e-Thr-Ile-Glu-Leu-Ser-Asn-Ile-Lys-Gl
u-Asn-Lys-Cys-Asn-Gly-Ala-Lys-Val-Ly
s-Leu-le-Lys-Gln-Glu-Leu-Asp-Lys-Ty
r-Asn-Ala-Val- (AEEA.MPA)) The preparation of maleimide peptides from peptides containing multiple protected functional groups and one cysteine is illustrated by the synthesis of modified RSV fusogenic peptides. The modified RSV fusogenic peptide was synthesized by C-terminal truncation by adding a lysine residue to the natural peptide sequence, as illustrated by the schematic diagram below. A cysteine residue is contained within the peptide sequence,
And if the peptide is essentially free of biological activity, this residue must be replaced with another amino acid (eg, alanine, methionine, etc.). In the synthesis below, cysteine (Cys) was added to the methionine (
Met).

[0328]   Solid-phase peptide of maleimide RSV fusogenic peptides on a scale of 100 μmol
Mode, manual solid phase synthesis and Fmoc protected Link Amide MBHA.
Symphony Peptide Synthesizer, Fmo
c-protected amino acid, O-benzotri in N, N-dimethylformamide (DMF)
Azol-1-yl-N, N, N ', N'-tetramethyl-uronium hexaf
Performed with Luorophosphate (HBTU), diisopropylethylamine
Activation with (DIEA) and piperidine deprotection of the Fmoc group (step 1
). Selective deprotection of the Lys (Aloc) group was carried out manually and 5 mL of C
HCl₃: NMM: HOAc (18: 1: 0.5) with 3 equivalents of Pd (
PPh₃)_FourThis is achieved by treating the resin with the solution of 2 hours (step 2). Next
Then, add this resin to CHCl₃(6 x 5 mL), 20% HOAc in DCM (
6 × 5 mL), DCM (6 × 5 mL), and DMF (6 × 5 mL)
. The synthesis is then automated for the addition of 3-maleimidopropionic acid.
(Step 3). Resin cleavage and product isolation was performed using 85% TFA / 5% TIS / 5.
% Thioanisole and 5% phenol, followed by dry
Et cooled by chair₂Precipitate with O (step 4). The product is Varia
n (Rainin) Preparation Binary HPLC System (: 30-55% B (H ₂ 0.045% TFA (A) and CH in O₃0.045% TFA in CN
(B)) Gradient elution) was used to remove the Phenomenex Luna 10μ solution.
Phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian
  Dynamax UVD II) (λ214 and 254 nm).
Purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min, RP-
The desired DAC is obtained in> 95% purity as determined by HPLC. these
The process is illustrated in the schematic diagram below.

[0329]

[Chemical 29] (3. Modification in internal amino acid) (Example 60 Cys-Asn-Leu-Lys-Glu-Asp-Gly-)
Modification of Gα Peptide in Ile-Ser-Ala-Ala-Lys-Asp-Val) The preparation of a maleimide peptide from a peptide containing multiple protected functional groups and one cysteine is illustrated by the synthesis of a modified Gα peptide. . The modified Gα peptide is synthesized by conjugation at internal amino acids as described below.

In the case where a cysteine residue is contained within the peptide sequence and is essentially free of peptide biological activity, this residue may be capped or another amino acid (eg alanine, methionine, etc.). ) Is replaced. Solid phase peptide synthesis of the modified Gα peptide on a 100 μmol scale was performed using manual solid phase synthesis and Fm.
Symphony Pept with oc protected Rink Amide MBHA
ide Synthesizer, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N in N, N-dimethylformamide (DMF).
Carried out with'-tetramethyl-uronium hexafluorophosphate (HBTU) and activated with N-methylmorpholine (NMM) to piperidine deprotect the Fmoc group (step 1). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0.5
This is achieved by treating the resin with a solution of 3 equivalents of Pd (PPh ₃ ) ₄ dissolved in 2) for 2 hours (step 2). The resin was then treated with CHCl ₃ (6 × 5 mL),
20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and D
Wash with MF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimidopropionic acid (step 3). Resin cleavage and product isolation
Performed with 5% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by precipitation with Et ₂ O cooled with dry ice (
Step 4). The product was subjected to Varian (Rainin) preparative binary HPLC.
System (: 30-55% B (0.045% TFA (A) and C in H ₂ O)
Using H ₃ CN 0.045% in TFA (B) gradient elution)), Pheno
menex Luna 10 [mu] Phenyl-hexyl, 21 mm x 25 cm column and UV detector (Varian Dynamax UVD II) ([lambda] 21.
4 and 254 nm) and purified by preparative reverse-phase HPLC at 9.5 mL / min for 180 min and> 95% as determined by RP-HPLC.
The desired DAC is obtained with purity.

E. Preparation of Modified Peptides from Peptides Containing Two Cysteines at a Disulfide Bridge If the peptides contain two cysteines as disulfide bonds, the peptides are supported prior to addition of maleimide. Cut from resin. We need to add Lys protected with an Mtt group in order to selectively deprotect lysine in the presence of other t-Boc protected lysines. All protecting groups are carboxy-terminal (which remains unprotected due to cleavage from the support resin)
And present with the exception of two cysteines, which need to be deprotected when the peptide is cleaved from the resin. Mild aerial oxidation gives a disulfide bridge, and the peptide can be purified at this stage. Liquid phase chemistry then needs to activate the C-terminus in the presence of the disulfide bridge and add a maleimide (via an amino-alkyl-maleimide) to the C-terminus. The peptide is then deprotected. Examples of therapeutic peptides containing two cysteines as disulfide bridges include human osteocalcin 1-49,
Human diabetes related peptides, human / dog atrial natriuretic peptide, bovine bactenecin, and human [Tyr0] -cortistatin 29.

Preparation of maleimide peptides from therapeutic peptides containing two cysteines in a disulfide bridge is exemplified by peptide synthesis as described below. The peptide may be modified at the N-terminus, the C-terminus, or the amino acids located between the N- and C-termini.

(1. Modification at N-Terminal) (Example 61 Modification of TH-1 Peptide at N-terminal. (Nε-MPA) N)
_{H 2 -Lys-Arg-Gly-} Asp-Ala-Cys-Glu-Gly-A
Preparation of sp-Ser-Gly-Gly-Pro-Phe-Cys) Contains multiple protected functional groups and no free cysteine residues (ie, all cysteine residues are constrained as disulfide bridges). The preparation of thiol-cyclized maleimide peptides from peptides is exemplified by the synthesis of modified TH-1 peptides.

Solid-phase peptide synthesis of the modified TH-1 peptide on a 100 μmol scale was performed manually, and Symphony Peptide Synthesizer, Fmoc with Fmoc-protected Rink Amide MBHA resin.
Protected amino acid, carried out with O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uronium hexafluorophosphate (HBTU) in N, N-dimethylformamide (DMF), N -Methylmorpholine (NMM)
To deactivate the Fmoc group by piperidine deprotection (step 1). Removal of the Acm group and resulting oxidation of the two Cys residues forming a cyclization on the resin DAC,
TI was achieved with (CF ₃ CO) ₂ (Step 2). Deprotection of the terminal Fmoc group,
Achieved with 20% piperidine, followed by coupling of 3-MPA (step 3). Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5.
Performed with% H ₂ O / 2% thioanisole and 2% phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 4). The product was applied to Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 2.
5 cm column and UV detector (Varian Dynamax UVD II
) (Λ 214 and 254 nm) with Dynamax C ₁₈ , 60Å, 8μ
Purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system using a m, 21 mm x 25 cm column. The product was a Hewlett Packard LCM equipped with a geode array detector.
R with S-1100 series spectrometer and with electrospray ionization
It had> 95% purity as determined by P-HPLC mass spectrometry. ESI-
_{MS m / z: C 66 H} 95 N 20 O 26 S 2 (MH +), 1646.8, Found: 164
6.7. These steps are illustrated in the schematic diagram below.

[0335]

[Chemical 30] Example 62 Synthesis of N-MPA-Ser ¹ -Somatostatin-28 Solid phase peptide synthesis of DAC: somatostatin-28 analogs on a 100 μmol scale was performed manually and with Symphony Peptide using Fmoc-protected Rink Amide MBHA resin. Run using Synthesizer. The following protected amino acids are sequentially added to the resin: Fmoc-Cy
s (Acm) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (
tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH
, Fmoc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, F
moc-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn (Trt)
-OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Acm) -O
H, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Lys (B
oc) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtB
u) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, F
moc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH, F
moc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Ser
(TBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Ala-O
H, Fmoc-Ser (tBu) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazole-1
Activation with -yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). A
Removal of the cm group and the resulting oxidation of the two Cys residues forming a disulfide bridge is accomplished with iodine (step 2). Deprotection of the terminal Fmoc group is achieved with 20% piperidine, followed by coupling of 3-MPA (step 3).
. Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5% H ₂ O.
/ Run with 2% thioanisole and 2% phenol, followed by precipitation by cold Et ₂ O in dry ice (Step 4). The product is Dyn
amax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax UVD II) (λ21
4 and 254 nm) Dynamax C ₁₈ , 60Å, 8 μm, 21 m
Binary H for Varian (Rainin) preparation using m × 25 cm column
Purify by preparative reverse phase HPLC using a PLC system to obtain the desired DAC in> 95% purity as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

[0336]

[Chemical 31] (2. Modification at C-Terminal) (Example 63 Synthesis of Somatostatin-28-EDA-MPA) Solid phase peptide synthesis of DAC: somatostatin-28 analog on a scale of 100 µmol was performed manually and SASRIN (super acid sensitive). Resin)
Execute on the Honey Peptide Synthesizer. The following protected amino acids are sequentially added to the resin: Fmoc-Cys (Acm) -OH.
Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fm
oc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (
Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH
, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-L
ys (Boc) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Gly
-OH, Fmoc-Ala-OH, Fmoc-Lys (Boc) -OH, Fmo
c-Arg (Pbf) -OH, Fmoc-Glu (OtBu) -OH, Fmoc
-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Ala-OH
, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc-Pro-OH
, Fmoc-Asn (Trt) -OH, Fmoc-Ser (tBu) -OH, F
moc-Asn (Trt) -OH, Fmoc-Ala-OH, Boc-Ser (
tBu) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ',
Activation with N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by removing 20% (V / V) piperidine in N, N-dimethylformamide (D
Activate with MF solution for 20 minutes (step 1). The fully protected peptide is cleaved from the resin with 1% TFA / DCM (step 2). Removal of the Acm group and the resulting oxidation of the two Cys residues forming a disulfide bridge is accomplished with iodine (step 3). Then ethylenediamine and 3-maleimidopropionic acid are sequentially added to the free C-terminus (step 4). The protecting groups are then cleaved and the product is loaded with 86% TFA / 5% TIS / 5% H ₂ O / 2.
% Thioanisole and 2% phenol, followed by precipitation with dry ice cooled Et ₂ O (step 5). The product is
Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × with ax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian Dynamax UVD II) (λ214 and 254 nm).
Binary HPL for Varian (Rainin) preparation using 25 cm column
Purify by preparative reverse phase HPLC using the C system to give the desired DAC in> 95% purity as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

[0337]

[Chemical 32] 3. Modifications at Internal Amino Acids Example 64 Synthesis of Lys ¹⁴ (ε-MPA) -Somatostatin-28 Solid phase peptide synthesis of DAC: somatostatin-28 analogs on a 100 μmol scale was performed manually and with Fmoc. Run on Symphony Peptide Synthesizer with protected Rink Amide MBHA resin. The following protected amino acids are sequentially added to the resin: Fmoc-Cys (
Acm) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tB
u) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, F
moc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmo
c-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -O
H, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Acm) -OH,
Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Lys (Alo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Glu (OtBu
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fm
oc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fm
oc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Ser (
tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Ala-OH
, Fmoc-Ser (tBu) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazole-1-
Activation with yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Ac
Removal of the m group and the resulting oxidation of the two Cys residues forming a disulfide bridge is accomplished with iodine (step 2). Selective deprotection of the Lys (Aloc) group was performed manually and 5 mL of CHCl ₃ : NMM: HOAc (18: 1: 0).
． This is accomplished by treating the resin with a solution of 3 equivalents of Pd (PPh3) 4 dissolved in 5) for 2 hours (step 3). This resin was then treated with CHCl ₃ (6 × 5 m
L), washed with 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 4). Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed by, Et ₂ cooled with dry ice
Precipitate with O (step 5). The product was treated with Dynamax C ₁₈ , 60Å, 8
μm guard module, 21 mm × 25 cm column and UV detector (Var
Ian Dynamax UVD II) (λ214 and 254 nm) and purified by preparative reverse-phase HPLC using a Varian (Rainin) preparative binary HPLC system with a Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column, RP- > 95 as determined by HPLC
The desired DAC is obtained in% purity. These steps are illustrated in the schematic diagram below.

[0338]

[Chemical 33] (F. Preparation of Modified Peptide from Peptide Containing Multiple Cysteines) (1. Modification at N-Terminal) (Example 65 N-MPA-Cys ¹ -Endothelin-1 (1-21) -OH
Synthesis of modified endothelin-1 analogs on a 100 μmol scale is performed manually and on a Symphony Peptide Synthesizer with Fmoc-protected Rink Amide MBHA resin. The following protected amino acids are sequentially added to the resin: Fmoc-Trp (B
oc) -OH, Fmoc-le-OH, Fmoc-le-OH, Fmoc-
Asp (OtBu) -OH, Fmoc-Leu-OH, Fmoc-His (Tr
t) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Phe-OH, F
moc-Tyr (tBu) -OH, Fmoc-Val-OH, Fmoc-Cys
(TBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Lys (
Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc-Met-O
H, Fmoc-Leu-OH, Fmoc-Ser (tBu) -OH, Fmoc-
Ser (tBu) -OH, Fmoc-Cys (tBu) -OH, Fmoc-Se
r (tBu) -OH, Fmoc-Cys (Acm) -OH. Let them be N, N-
Dissolve in dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DI.
Activate with EA). Removal of the Fmoc protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Two Cys to remove Acm group and form disulfide bridge
The resulting oxidation of the residue is accomplished with iodine (step 2). Removal of the tBu group and resulting oxidation of the other two Cys forming a second disulfide bridge on the resin is accomplished with thallium (III) trifluoroacetate (step 3).
. Deprotection of the terminal Fmoc group was performed with 20% piperidine followed by 3-MPA.
(Step 4). Resin cleavage and product isolation
6% TFA / 5% TIS / 5% H ₂ O / 2% thioanisole and 2%
Of phenol, followed by precipitation with Et ₂ O cooled with dry ice (step 5). The product was loaded onto a Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (Varian).
Dy with Dynamax UVD II) (λ214 and 254 nm)
Var using a namax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column
Purify by preparative reverse phase HPLC using an ian (Rainin) preparative binary HPLC system to obtain the desired DAC in> 95% purity as determined by RP-HPLC.

[0339]

[Chemical 34] These steps are illustrated in the schematic diagram above.

[0340] (Modification at 2.C end) (Example 66 endothelin ^{-1 (1-21) Lys 22 - (} Nε-MPA) -
Synthesis of OH) Solid phase peptide synthesis of modified endothelin-1 analogs on a 100 μmol scale is performed manually and on a Symphony Peptide Synthesizer with Fmoc-protected Rink Amide MBHA resin. The following protected amino acids are continuously added to the resin: Fmoc-Lys (Al
oc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-le-OH,
Fmoc-le-OH, Fmoc-Asp (OtBu) -OH, Fmoc-L
eu-OH, Fmoc-His (Trt) -OH, Fmoc-Cys (Acm)
-OH, Fmoc-Phe-OH, Fmoc-Tyr (tBu) -OH, Fmo
c-Val-OH, Fmoc-Cys (tBu) -OH, Fmoc-Glu (O
tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (Ot
Bu) -OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-.
Ser (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Cy
s (tBu) -OH, Fmoc-Ser (tBu) -OH, Boc-Cys (A
cm) -OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ', N
Activation with'-tetramethyl-uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA). Removal of the Fmoc protecting group was accomplished by removing 20% (V / V) piperidine in N, N-dimethylformamide (DM).
F) Activate with solution for 20 minutes (step 1). Removal of the Acm group and the resulting oxidation of the first two Cys residues forming the first disulfide bridge on the resin is accomplished with iodine (step 2). Removal of the tBu group and the resulting oxidation of the other two Cys to form a second disulfide bridge on the resin was carried out by thallium (III
) Achieved with trifluoroacetate (step 3). Selective deprotection of the Lys (Aloc) group was carried out manually and 5 mL of CHCl ₃ : NMM: HOA
This is accomplished by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in c (18: 1: 0.5) for 2 hours (step 4). Then, this resin is treated with CHC
_{l 3 (6 × 5mL),} 20% HOAc (6 × 5mL) in DCM, DCM (6
X 5 mL), and DMF (6 x 5 mL). This synthesis is then
Re-automating for the addition of maleimido prepionic acid (step 5). Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed by, Et ₂ cooled with dry ice Precipitate with O (step 6). The _{product, Dynamax C 1 8, 60Å,} 8μm guard module, 21 mm × 25 cm column and UV
Detector (Varian Dynamax UVD II) (λ 214 and 25
Dynamax C ₁₈ , 60 Å, 8 μm, 21 mm x 25 cm with 4 nm)
Purify by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system with a column to obtain the desired DAC in> 95% purity as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

[0341]

[Chemical 35] (3. Modification in Internal Amino Acid) (Example 67 Lys ⁴ (Nε-MPA) Saraphotoxin B (1-21)-
Synthesis of OH) Solid phase peptide synthesis of the modified Sarafotoxin-B analog on a 100 μmol scale is performed manually and on a Symphony Peptide Synthesizer with Fmoc-protected Rink Amide MBHA resin. The following protected amino acids are sequentially added to the resin: Fmoc-Trp (
Boc) -OH, Fmoc-le-OH, Fmoc-Val-OH, Fmoc
-Asp (OtBu) -OH, Fmoc-Gln (Trt) -OH, Fmoc-
His (Trt) -OH, Fmoc-Cys (Acm) -OH, Fmoc-Ph
e-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Leu-OH, Fm
oc-Cys (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmo
c-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Thr (tBu) -OH, Fmoc-Met-OH, Fmoc-Asp (Ot
Bu) -OH, Fmoc-Lys (Aloc) -OH, Fmoc-Cys (tB
u) -OH, Fmoc-Ser (tBu) -OH, Boc-Cys (Acm)-
OH. They are dissolved in N, N-dimethylformamide (DMF) and according to the sequence O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uronium hexafluorophosphate (HBTU). ) And diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is activated with 20% (V / V) piperidine in N, N-dimethylformamide (DMF) for 20 minutes (step 1). Removal of the Acm group and the resulting oxidation of the first two Cys residues forming the first disulfide bridge on the resin is accomplished with iodine (step 2). Removal of the tBu group and resulting oxidation of the other two Cys forming a second disulfide bridge on the resin is accomplished with thallium (III) trifluoroacetate (step 3). Selective deprotection of the Lys (Aloc) group was performed manually, and 5 mL of CHCl ₃ : NMM: HOAc (18
This is achieved by treating the resin with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in 1: 0.5) for 2 hours (step 4). The resin was then treated with CHCl ₃ (6
X5 mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL
), And DMF (6 × 5 mL). The synthesis is then re-automated for the addition of 3-maleimido prepionic acid (step 5). Resin cleavage and product isolation was performed using 86% TFA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed by, Et ₂ cooled with dry ice Precipitate with O (step 6). The product was converted to Dynamax C ₁₈ , 60
Å, 8 μm guard module, 21 mm × 25 cm column and UV detector (
Varian Dynamax UVD II) (λ214 and 254 nm)
Purified by preparative reverse-phase HPLC using a Varian (Rainin) preparative binary HPLC system using a Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column equipped with, as determined by RP-HPLC,
The desired DAC is obtained with> 95% purity. These steps are illustrated in the schematic diagram below.

[0342]

[Chemical 36] F. Peptide Stability Assay Example 68 K5 Peptide Stability Assay A peptide stability assay was performed. (MPA) -Pro-Arg-Lys-
Leu-Tyr-Asp-Lys- NH 2. 2TFA was synthesized as above,
And MPA-K5 was identified. Unmodified corresponding peptide Pro-Arg-Lys
-Leu-Tyr-Asp-Lys was also prepared in Example 20 without the addition of 3-MPA.
And was identified as K5.

K5 (MW 1260.18, 918.12 free base) was prepared as a 100 mM stock solution in water. MPA-K5 (MW = 1411.17, 1069.1)
1 free base) was prepared as a 100 mM stock solution in water. Human Serum Albumin (HSA) Alb available from Alpha Therapeutic
25% solution as utein® (calculated 250 mg / ml, 3.75
mM). Human plasma from Golden West Biologica
Obtained from Is.

(1) Stability of K5 in Human Plasma K5 was prepared as a 1 μM solution and dissolved in 25% human serum albumin. This mixture is then subjected to a final concentration of 160 mM K in the presence of human plasma.
Incubated up to 5 at 37 ° C. Aliquots of 100 μl were collected from plasma at 0, 4 and 24 hours. A 100 μl aliquot was added to 100 μl of blocking solution (5 volumes of 5% Z to precipitate all proteins).
It was mixed with NSO _4/3 volumes of acetonitrile / 2 volumes of methanol). The sample was centrifuged at 10,000 g for 5 minutes, and the supernatant containing the peptide was
It was reconverted and filtered through a 0.22 μm filter. The presence of free intact K5 peptide was assayed by HPLC / MS. The results are shown below. The HPLC parameters for detection of K5 peptide in serum were as follows.

The HPLC method was as follows: A Vydac C18 250 × 4.
． A 6 mm, 5μ particle size column was utilized. The column temperature was 30 ° C. and the flow rate was 0.5 ml / min. Flowable phase A was 0.1% TFA / water. Flowable phase B was 0.1% TFA / acetonitrile. Injection volume is 10
It was μl.

The gradient was as follows: Time (min)% A% B 0 95 5 20 70 30 25 25 10 90 30 30 10 90 35 35 95 5 45 95 5 protein detected at 214, 215 and 334 nm. . For mass spectrometric analysis, the ionization mode was API-electrospray (positive mode) in the M / Z range 300-2000. The gain was 3.0, the fragmentor was 120v, the threshold was 20, and the step size was 0.1. The gas temperature was 350 ° C. and the dry gas volume was 10.0 l / min. Neb pressure was 24 psi and Vcap was 3500V. The HPLC method was as follows: A Vydac C18 250 × 4.
． A 6 minute, 5μ particle size column was utilized. The column temperature was 30 ° C. and the flow rate was 0.5 ml / min. Flowable phase A was 0.1% TFA / water. Mobile Phase B was 0.1% TFA / acetonitrile. Injection volume is 1
It was 0 μl.

The gradient was as follows: Time (min)% A% B 0 95 5 20 70 30 25 25 10 90 90 30 10 90 90 35 95 5 45 95 5 protein detected at 214, 254 and 334 nm. did. For mass spectrometric analysis, the ionization mode was API-electrospray (positive mode) in the M / Z range 300-2000. The gain was 3.0, the fragmentor was 120v, the threshold was 20, and the step size was 0.1. The gas temperature was 350 ° C. and the dry gas volume was 10.0 l / min. Neb pressure was 24 psi and Vcap was 3500V.

Times% K5 peptide in plasma 0 hours 100% 4 hours 9% 24 hours 0% The results demonstrate that the unmodified K5 peptide is unstable in plasma as well as the result of protease activity. .

(2) Stability of MPA-K5-HSA conjugate in plasma MPA-K5 (modified K5 peptide) was incubated for 2 hours with 25% HSA at room temperature. The MPA-K5-conjugate was then added to a final concentration of 160 μm.
At 37 ° C. in the presence of human plasma. After the specified incubation period (0, 4 and 24 hours), 100 μl aliquots were taken and filtered through a 0.22μ filter. The existence of an intact conjugate is
Assayed by PLC-MS.

The column was an Aquapore RP-300, 250 x 4.6 mm, 7μ particle size. The column temperature was 50 ° C. Liquid phase A is 0.1% T
FA / water. Flowable phase B was 0.1% TFA / acetonitrile. The injection volume was 1 μl. The gradient was as follows: Time (min)% A% B Flow rate (ml / min) 0 66 34 0.700 1 66 34 0.700 25 58.8 41.2 0.700 30 50 50 0 .70 35 5 95 1.00 41 5 95 1.00 45 45 66 34 1.00 46 66 34 0.70.

Peptides were detected at 214 nm for quantification. For mass spectrometric analysis of peptides, the ionization mode is API-electrospray in the range 1280-1500 m / z, gain 1.0, fragmentor 125 V, threshold 100,
The step size was 0.40. The gas temperature is 350 ° C and the dry gas is
It was 13.0 l / min. Pressure is 60 psi and Vcap is 60
It was 00V. The results are shown below.

Approximately 33% of circulating albumin in the bloodstream is mercaptoalbumin (
SH-albumin), which is blocked by endogenous sulfhydryl compounds (eg cysteine or glutathione) and is therefore available for reaction with maleimide groups. The remaining 66% of circulating albumin is capped or blocked by sulfhydryl compounds. The HPLC MS assay allows the identification of capped HSA, SH-albumin, and K5-MPA-albumin. MPA covalently binds to a free thiol on albumin. The stability of the three forms of albumin in plasma is shown below.

[0353]

[Table 1] The percentage of K5-MPA-HSA remained relatively constant throughout the 24-hour plasma assay, symmetrically with unmodified K5, up to 9% of the original amount of K5 in plasma only at 4 hours. Reduced. The results show that, in contrast to K5, which is quite unstable in plasma,
K5-MPA-HSA demonstrates considerable stability in plasma from peptidase activity.

Example 70 Dynorphin Peptide Stability Assay To determine the stability of peptide conjugates in the presence of serum peptidase, Dy
n A- (1-13) -OH, Dyn A- (1-13) -NH ₂ and Dyn
The serum stability of A 1-13 (MPA) -NH _2, were compared. Dyn A- (
1-13) -OH, Dyn A- (1-13) -NH ₂ and Dyn A-1-
13 (MPA) -NH ₂ were synthesized as described above. Dynorphin peptide,
Human heparinized plasma was used to mix to a final concentration of 4 mg / mL. Required incubation time at 37 ° C (0, 20, 20, 60, 120, 180, 3
After 60 and 480 minutes) a 100 μL aliquot was added to 100 μL of a block solution (5 volumes of 5% ZnSO _{4 in} water, 3 volumes of acetonitrile, 2 volumes of methanol) to precipitate all proteins. Centrifuge (10,000
g, 2.5 min), the clear supernatant was collected, filtered through a 0.45 μm filter and stored on ice until LC / MS analysis.

Samples were analyzed by LC at 214 nm to detect the presence of different compounds and MS to determine the identity of the detected compounds. Then LC
The integrated area% for each peak from the chromatogram was plotted against time and relative stability determined in human plasma.

Stability data for Dyn A- (1-13) -OH and Dyn A- (1-13) -NH ₂ were in agreement with those reported in the literature: Proteolytic degradation of the dynorphin peptide. Is pretty quick. Dyn A- (1-13
) -OH had a half-life of approximately 10 minutes. Dyn A- (1-13) -NH ₂ had a half-life of about 30 minutes. In contrast, Dyn A- (1-13) -NH ₂ showed marked stability in the presence of serum peptidase activity. The unmodified dynorphin peptide is degraded within 60 minutes. In contrast, the modified dynorphin peptide (Dyn A- (1-13) -NH ₂ ) is stable in serum peptidase activity up to 480 minutes.

Stability determinations of dynorphin conjugates are determined by ELISA. Whether the observed signal is due to the dynorphin conjugate and this conjugate is
LC mass spectroscopic analysis of the reaction mixture after 8 hours was performed to determine something.
The use of mass spectroscopy allows the molecular weight of the conjugate to be determined, and whether it is any truncated form of the dynorphin conjugate. Mass spectrometry of human plasma shows two forms of albumin, a free thiol at 66436 Da and an oxidized form at 66557 Da. Mass spectroscopy also showed Dyn 1 intact with Dyn 2-13 cleaved conjugate (68046 Da) in an equal mixture.
A distinction can be made between the -13 conjugate (68207 Da).

After 480 minutes of exposure, mass spectrometric analysis of dynorphin samples obtained from serum for serum peptidases identified only the presence of intact conjugate (68192 Da) and not the presence of decay products, Demonstrates the stability of the dynorphin conjugate from serum peptidase activity.

[0359]

[Table 2]

[Sequence list]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) // A61K 38/00 A61K 37/02 (31) Priority claim number 60 / 159,783 (32) Priority date October 15, 1999 (October 15, 1999) (33) Priority claiming countries United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, A , AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN , MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Milner, Peter G. United States California 94022, Los Altos Hills, Manuela Road 14690 (72) Inventor Holmes, Darren El. Canada H3 G1 Tea3 Quebec, Montreal, Drummond Street 3450 (72) Inventor Ciboude, Karen Canada H3 W2 L5 Quebec, Montreal, Number 407, Bonavista Street 4700 F-term (reference) 4C076 CC30 EE41Q EE59Q 4C084 AA03 DC50 NA03 NA14 ZC031 4H045 AA20 BA12 BA13 BA14 BA15 BA16 BA17 BA18 BA19 BA41 BA50 CA42 EA20 FA33

Claims (19)

[Claims] 1. A method of synthesizing a peptidase-stabilized therapeutic peptide containing between 3 and 50 amino acids, said peptide comprising a carboxy terminal amino acid, an amino terminal amino acid, a therapeutically active region. And having a therapeutically lower active region, the method comprises the steps of: a) synthesizing the peptide from the carboxy-terminal amino acid when the therapeutic peptide does not contain cysteine and adding a reactive group A step of adding to the carboxy terminal amino acid, or adding a terminal lysine to the carboxy terminal amino acid and adding the reactive group to the terminal lysine, wherein the reactive group is an amino group on a blood component, A step capable of reacting with a hydroxyl group or a thiol group to form a stable covalent bond; or b) when the therapeutic peptide comprises only cysteine, Reacting the cysteine with a protecting group prior to adding a reactive group to the amino acid in the therapeutically less active region; or c) if the therapeutic peptide contains two cysteines as a disulfide bridge, Oxidizes two cysteines and attaches the reactive group to the amino terminal amino acid,
Or adding to the carboxy-terminal amino acid, or to an amino acid located between the carboxy-terminal amino acid and the amino-terminal amino acid; or d) when the therapeutic peptide contains more than 2 cysteines as disulfide bridges, Serially oxidizing the cysteines in the disulfide bridge and purifying the peptide prior to addition of the reactive group to the carboxy-terminal amino acid. 2. The method of claim 1, wherein the reactive group is attached to the therapeutic peptide via a linking group. 3. The method of claim 1, wherein the blood component is albumin. 4. The method of claim 3, wherein the reactive group comprises a maleimide. 5. A method for protecting a therapeutic peptide from peptidase activity in vivo, said peptide comprising between 3 and 50 amino acids and carboxy-terminal and amino-terminal and carboxy-terminal amino acids. And having an amino terminal amino acid, the method comprises: a) attaching a reactive group to the carboxy terminal amino acid, the amino terminal amino acid, or an amino acid located between the amino terminal amino acid and the carboxy terminal amino acid. Modifying the peptide, whereby the reactive group is capable of forming a covalent bond in vivo with a reactive functional group on a blood component; and b) on the blood component with the reactive group. Forming a modified peptide-blood component conjugate by forming a covalent bond with the reactive functional group of the peptidase. Step of protecting the peptide from the activity; encompasses a method. 6. The method further comprising the step of administering said modified peptide in vivo prior to step (b), such that said modified peptide-blood component conjugate is formed in vivo. The method according to 5. 7. The method of claim 5, wherein step (b) occurs ex vivo. 8. The method of claim 5, wherein the reactive group is a maleimide group. 9. The method of claim 5, wherein the reactive group is attached to the peptide via a linking group. 10. The method of claim 8, wherein the blood component is albumin. 11. The method of claim 5, wherein the one or more amino acids is synthetic. 12. A method for the protection of a therapeutic peptide in vivo from peptidase activity, said peptide containing between 3 and 50 amino acids and comprising a therapeutically active region of amino acids and Having a therapeutically less active region of an amino acid, the method comprising: a) determining the therapeutically active region of the amino acid; b) at an amino acid contained in the therapeutically less active region. Modifying the peptide by attaching a reactive group to the amino acid to form a modified peptide, such that the modified peptide is a process having therapeutic activity, wherein the reactive group is Can form a covalent bond in vivo with a reactive functional group on the blood component; and c) forms a covalent bond between the reactive entity and the reactive functional group on the blood component to form the peptide. -Forming a blood component conjugate, thereby protecting the peptide from peptidase activity. 13. The method of claim 12, further comprising the step of administering the modified peptide in vivo prior to step (c) such that the peptide-blood component conjugate is formed in vivo. The method described. 14. The method of claim 12, wherein step (c) occurs ex vivo. 15. The method of claim 12, wherein the peptide has a carboxy terminus, an amino terminus, a carboxy terminus amino acid, and an amino terminus amino acid, and wherein step (b) comprises And further: a) modifying the peptide at the carboxy-terminal amino acid of the peptide if the therapeutically less active region is located at the carboxy terminus of the peptide; or b) the lower activity. A region of is located at the amino terminus of the peptide, modifying the peptide at the amino terminal amino acid of the peptide; or c) the less active region portion is also at the amino terminus of the peptide. If it is also not located at the carboxy terminus, the peptide is placed at an amino acid located between the carboxy terminus and the amino terminus. Modifying step; 16. The method according to claim 12, wherein the reactive group is a maleimide group. 17. The method of claim 12, wherein the reactive group is attached to the peptide via a linking group. 18. The method of claim 12, wherein the blood component is albumin. 19. The method of claim 12, wherein the one or more amino acids is synthetic.